Insulin resistance and dyslipidaemia associated with smoking may result from increased secretion of anti-insulin hormones. We compared the pituitary-adrenocortical function using oral glucose tolerance, dexamethasone suppression and ACTH stimulation tests in smoking (n = 22) and non-smoking (n = 22) healthy males matched for age, body mass index, and waist-to-hip ratio. Smokers had lower HDL-cholesterol (p < 0.02), and higher triglyceride (p < 0.001), basal cortisol (p < 0.05), insulin (p < 0.05), and C-peptide (p < 0.02) levels, and a higher response of insulin and C-peptide to oral glucose (p < 0.005) than non-smokers, while the ACTH, cortisol, 17-hydroxyprogesterone, dehydroepiandrosterone, and androstenedione responses to oral glucose were similar in both groups. No differences were found in the response of cortisol, 17-hydroxyprogesterone, dehydroepiandrosterone, and androstenedione to dexamethasone. In contrast, the response of 17-hydroxyprogesterone (p = 0.04), dehydroepiandrosterone (p = 0.007), and androstenedione (p = 0.001) to ACTH was higher in smokers than non-smokers, while the increase in cortisol was of marginal significance (p = 0.07). In multiple regression analyses the dehydroepiandrosterone response to ACTH was a significant determinant of insulin, C-peptide, and triglyceride levels independent of physical activity, waist-to-hip ratio and HDL-cholesterol. Thus, smoking inhibits the adrenal 21-hydroxylase resulting in an increase in the production of adrenal androgens, which might contribute to the insulin resistance and dyslipidaemia in smokers.