It is well established that the intracellular receptors of androgens act as transcription factors upon their activation by androgen binding. However, a growing number of studies have associated androgens with rapid biological responses independent of their classical action mechanism. In this sense, 5α- and 5β-dihydrotestosterone elicited a rapid positive inotropism in the isolated left atrium of the rat via cAMP-dependent mechanisms that may involve genomic effects. In addition, polyamines are mediators of several biological actions including those acute and long-term effects induced by androgens in the heart. The present study analyzed the role of polyamine synthesis in the cardiotonic effect of androgens in the left atrium of male Wistar rats, electrically stimulated (0.5 Hz, 5 ms and supramaximal voltage) and placed in an organ bath in 10 ml of Tyrode's solution. Incubation in the organ bath with an inhibitor of ornithine decarboxylase activity, α-difluoromethylornithine 10 mM, significantly decreased the positive inotropism induced by 5α- and 5β-dihydrotestosterone (0.1–100 μM). This suggests that ornithine decarboxylase seems to be involved in androgen-induced positive inotropism. Furthermore, 6-min exposure to 5α- or 5β-dihydrotestosterone significantly increased the activity of ornithine decarboxylase from 61.81±7.53 (control) to 93.28±9.45 and 80.28±12 pmol/h/mg of protein, respectively. Northern blot analysis showed that 5α- and 5β-dihydrotestosterone did not modify the level of expression of the ornithine decarboxylase gene. Therefore, our results suggest that polyamine synthesis might be involved in the positive inotropism elicited by androgens through the stimulation of ornithine decarboxylase activity without changes in the expression of the ornithine decarboxylase gene.