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Background: Substance use, particularly binge drinking of alcohol and noninjection substance use, is associated with increased risk for HIV infection among youth, but structured substance use screening and brief intervention are not often provided as part of HIV risk reduction., Objective: The purpose of the study was to test the efficacy of a fully automated electronic screening and brief intervention, called Step Up, Test Up, to reduce alcohol misuse among adolescents and young adults presenting for HIV testing. Secondary objectives were reduction in sexual risk and uptake of pre-exposure prophylaxis (PrEP) for HIV prevention., Methods: Youth aged 16 years to 25 years who presented for HIV testing at community-based locations were recruited for study participation. Those who screened at moderate to high risk on the Alcohol Use Disorders Identification Test were randomized (1:1) to either an electronic brief intervention or a time-attention control. The primary outcome was change in alcohol use at 1, 3, 6, and 12-month follow-ups. Negative binomial and log binomial regression analyses with generalized estimating equations were conducted to evaluate the intervention efficacy., Results: Among a sample of 329 youth, there were no significant differences in alcohol use outcomes between conditions over time or at the 1, 3, 6, or 12-month time points. In terms of secondary outcomes, there was evidence of reduction in condomless insertive anal sex under the influence of alcohol and drugs at 12 months compared with 3 months in the intervention versus the attention control condition (incidence rate ratio=0.15, 95% CI 0.05-0.44); however, there were no other significant differences in sexual risk and no difference in PrEP engagement., Conclusions: We found no effect of electronic brief intervention to reduce alcohol use and some effect on sexual risk among youth aged 16 years to 25 years who present for HIV testing., Trial Registration: ClinicalTrials.gov number NCT02703116; https://clinicaltrials.gov/ct2/show/NCT02703116., International Registered Report Identifier (irrid): RR2-10.1186/s12889-020-8154-6., (©Niranjan S Karnik, Lisa M Kuhns, Anna L Hotton, Natascha Del Vecchio, Moira McNulty, John Schneider, Geri Donenberg, Kristin Keglovitz Baker, Rose Diskin, Abigail Muldoon, Juan Rivera, Faith Summersett Williams, Robert Garofalo. Originally published in JMIR Mental Health (https://mental.jmir.org), 29.03.2023.)