The effect of smoking on androgen levels is important given the recent interest in the link between low levels of androgens and the development of cardiovascular disease. Numerous studies examining the effects of cigarette smoking on the levels of total and free testosterone have reported conflicting findings, but there has been no accurate assessment of the effects of cigarette smoking on the levels of bioavailable testosterone [not bound to sex hormone-binding globulin (SHBG)]. We attempted to determine whether smoking affects the level of bioavailable testosterone. We undertook a case-control study of 25 healthy male smokers and 25 healthy never-smokers, matched by age and body mass index. Early morning levels of total, free and bioavailable testosterone, 17β-oestradiol, SHBG and cotinine were determined and compared between the two groups. Levels of total (18.5 ± 4.6 ± nM versus 15.1 ± 4.9 ± nM, P = 0.01) and free testosterone (462 ± 91 ± pM versus 402 ± 93 ± pM, P = 0.03) were found to be higher in smokers compared with non-smokers respectively, as was SHBG (34.1 ± 12.8 versus 28.1 ± 9.0 ± nM, P = 0.06). There were no significant differences in the levels of bioavailable testosterone (3.78 ± 1.59 versus 3.51 ± 1.26 ± nM, P = 0.49) or 17β-oestradiol (44.5 ± 11.4 versus 42.3 ± 11.5 ± pM, P = 0.50) between smokers and non-smokers respectively. These data suggest that cigarette smoking has no significant effect on the biologically active fraction of testosterone, but may influence the levels of total and free testosterone through changes in the levels of SHBG.