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Objective This study aimed to investigate the possibility of a prospective association between long working hours and use of psychotropic medicine. Methods Survey data drawn from random samples of the general working population of Denmark in the time period 1995-2010 were linked to national registers covering all inhabitants. The participants were followed for first occurrence of redeemed prescriptions for psychotropic medicine. The primary analysis included 25 959 observations (19 259 persons) and yielded a total of 2914 new cases of psychotropic drug use in 99 018 person-years at risk. Poisson regression was used to model incidence rates of redeemed prescriptions for psychotropic medicine as a function of working hours (32-40, 41-48, >48 hours/week). The analysis was controlled for gender, age, sample, shift work, and socioeconomic status. A likelihood ratio test was used to test the null hypothesis, which stated that the incidence rates were independent of weekly working hours. Results The likelihood ratio test did not reject the null hypothesis (P=0.085). The rate ratio (RR) was 1.04 [95% confidence interval (95% CI) 0.94-1.15] for the contrast 41-48 versus 32-40 work hours/week and 1.15 (95% CI 1.02-1.30) for >48 versus 32-40 hours/week. None of the rate ratios that were estimated in the present study were statistically significant after adjustment for multiple testing. However, stratified analyses, in which 30 RR were estimated, generated the hypothesis that overtime work (>48 hours/week) might be associated with an increased risk among night or shift workers (RR=1.51, 95% CI 1.15-1.98). Conclusion The present study did not find a statistically significant association between long working hours and incidence of psychotropic drug usage among Danish employees. [ABSTRACT FROM AUTHOR]