Background Adenylyl cyclase (AC) is an enzyme that can regulate the physiologic effects of numerous drugs and hormones through the production of cyclic adenosine-3′,5′-monophosphate (cAMP). Some studies suggest that certain measures of AC activity are lower among depressed subjects. We examined the relationship between various measures of AC activity and major depression, taking into account potential confounders, such as drug use and gender. Methods We assessed the relationship between platelet levels of AC activity and lifetime diagnosis of major depression among 1481 participants (226 subjects with a history of major depression and 1255 control subjects) in an international, cross-sectional study initiated by the World Health Organization and the International Society on Biomedical Research on Alcoholism. Results After accounting for recent drug use, subjects with a history of major depression had markedly lower mean levels for all measures of platelet AC activity compared with control subjects. The adjusted odds ratios for major depression comparing the bottom to the top quartile of AC activity were 2.69 for basal (95% confidence interval [CI] 1.30–5.56), 3.72 for cesium fluoride-stimulated (95% CI 1.54–8.98), 6.20 for forskolin-stimulated (95% CI 2.04–18.80), and 2.20 for Gpp(NH)p-stimulated (95% CI 1.03–4.70). Conclusions Subjects with major depression have lower platelet AC activity levels, and this relationship is dramatically attenuated by various types of drug use.