Androgens relax several smooth muscles, including the airways. They also contract ileum and myocardium via nongenomic mechanisms. To find out whether androgens modulate airway smooth muscles in different species and further assess their mechanism of action, regarding the role of β-adrenoceptors, polyamines and extracellular Ca2+, and the modulation of contraction, 5α-dihydrotestosterone, testosterone and 5β-dihydrotestosterone were used. A preliminary study was performed to evaluate the effect of 5α-dihydrotestosterone, a non-aromatisable derivate of testosterone, in isolated guinea-pig trachea and a more exhaustive characterisation was followed in bovine trachea, to also characterise the effect of testosterone and 5β-dihydrotestosterone. The androgens elicited a nongenomic epithelium-independent relaxation of the trachea which had been precontracted. In the bovine trachea, the order of potency was: testosterone > 5α-dihydrotestosterone = 5β-dihydrotestosterone. This effect was inversely proportional to the magnitude of carbachol-raised tone and was independent of β2-adrenoceptors, since the β-blockers, propranolol and ICI-118,551, and β2-adrenoceptor desensitisation did not modify 5α-dihydrotestosterone-elicited relaxation. 5α-Dihydrotestosterone was unable to displace the radiolabel, [3H]dihydroalprenolol, from these receptors in the binding assay. Polyamine synthesis was not involved in this androgen effect, since an ornithine decarboxylase inhibitor, α-difluoromethylornithine, was ineffective. The androgens were more effective relaxing bovine trachea precontracted by KCl (80 mM), suggesting a calcium entry blockade, as reported for several smooth muscles. This mechanism might be involved in the observed 5α-dihydrotestosterone facilitation of salbutamol-relaxation. Androgens facilitated carbachol-elicited contraction independently of polyamine synthesis, contrary to what has been reported in the ileum. Therefore, androgens modulate tracheal smooth muscle tone which might be of importance in the regulation of airway reactivity.