Background: Aedes (Stegomyia) albopictus (Skuse) impacts human outdoor activity because of its aggressive biting behavior, and as a major vector of mosquito-borne diseases, it is also of public health importance. Although most mosquito species exhibit crepuscular activity by primarily host seeking at dawn and dusk, Ae. albopictus has been traditionally characterized as a diurnal or day-biting mosquito. With the global expansion and increased involvement of Ae. albopictus in mosquito-borne diseases, it is imperative to elucidate the diel activity of this species, particularly in newly invaded areas. Methodology and principal findings: Human sweep netting and carbon dioxide-baited rotator traps were used to evaluate the diel activity of Ae. albopictus in two study sites. Both trapping methods were used in New Jersey's Mercer County, USA (temperate/urban), while only human sweep netting was used in Florida's Volusia County, USA (subtropical/suburban). Human sweep netting was performed to determine adult mosquito activity at Sunrise, Solar Noon, Sunset, and Lunar Midnight. Because New Jersey is in a temperate area, diel activity was investigated during the early season (3–19 July), peak season (25 July-19 September), and late season (22 September- 22 October). Aedes albopictus showed the highest activity during peak and late seasons at Solar Noon (P < 0.05). At Sunrise and Sunset during the peak season, Ae. albopictus activity was similar. Lunar Midnight activity was significantly lower than Sunrise and Solar Noon (P < 0.05) but was similar to that of Sunset. In the late season, the highest activity was observed during Solar Noon while the least activity was observed during Sunrise and Lunar Midnight (P<0.05). Bottle rotator traps used in conjunction with the human sweep net technique exhibited similar results. Seasonal activity was not differentiated in Florida due to the consistent subtropical climate. The highest adult activity was observed at Sunrise using human sweep netting, but it was not significantly different from Solar Noon and Sunset. The lowest adult activity was observed at Lunar Midnight; however, it was not significantly different from Solar Noon and Sunset. These results provide evidence that the diel activity of Ae. albopictus, contrary to the common perception of its diurnal activity, is much more varied. Conclusion/Significance: Involvement of Ae. albopictus in the transmission of debilitating mosquito-borne pathogens such as chikungunya, dengue, and Zika virus, coupled with its affinity to thrive in human peridomestic environments, substantiates that our findings have global implications in areas where Ae. albopictus populations established. It also highlights the importance of behavioral studies of vector species which will not only help mosquito control professionals plan the timing of their control efforts but also provides empirical evidence against conventional wisdoms that may unjustly persist within public health stewards. Author summary: The Asian tiger mosquito, Aedes albopictus, is an invasive mosquito which is now established in at least 40 states in the USA. Lack of efficient surveillance and control methods against Ae. albopictus, in addition to human-aided accidental transportations, have played a great role in its rapid expansion. Although surveillance measures are becoming more systematic and effective, control of this species still poses a great challenge. Aedes albopictus is difficult to control in the larval stage because it primarily develops in artificial containers that are widespread in peridomestic habitats. These habitats are not only ubiquitous in these environments, they are also cryptic, inaccessible, and extremely difficult to control. Therefore, control of Ae. albopictus in these environments often relies on adult control measures which utilize insecticides dispersed through ultra-low volume equipment as a cold aerosol space spray. These adulticide applications are often conducted at night against endemic mosquito species which are primarily active between dawn and dusk. However, since Ae. albopictus has been traditionally classified as a day-biting mosquito, mosquito control specialists have had doubts about the efficacy of a nocturnal application against a diurnally active mosquito. These uncertainties about intervention efforts become even more important during public health outbreaks of mosquito-borne pathogens such as chikungunya, dengue, or Zika viruses when protection of public health is of paramount importance in peridomestic habitats. Our investigations provide evidence that Ae. albopictus exhibits activity throughout the day and night and that nighttime adulticide applications may indeed be effective against this species, and should not be disregarded. [ABSTRACT FROM AUTHOR]