We demonstrate that testosterone and its active metabolite 5α-dihydrotestosterone acutely (∼30 min) potentiate mouse ileal, but not duodenal, muscle activity. Androgens augment the amplitude of spontaneous peak-to-peak oscillations, alter the spontaneous activity frequency spectrum, and increase the amplitude of calcium-induced and carbachol-induced contractions. Concentration-dependence analyses revealed that maximal potentiation (449–910%) occurred at physiological concentrations of androgens (100 pm to 10 nm) with EC₅₀ values in the picomolar range (8–20 pm). Western blot analyses using an antiandrogen receptor (anti-AR) antibody revealed the presence of two different AR proteins migrating at 87 and 110 kDa in ileal, but not duodenal, extracts. Androgen-induced potentiation was prevented by preincubation with AR antagonists flutamide or cyproterone acetate but was unaffected by pretreatment with cycloheximide plus actinomycin D, indicating that potentiation was mediated by ARs via a novel nongenomic mechanism. Androgen effects were mimicked by polyamines putrescine and spermine and were blocked by the ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase inhibitors α-difluoromethylornithine and berenil, respectively. Accordingly, androgens increase α-difluoromethylornithine-sensitive ornithine-decarboxylase- mediated l-ornithine decarboxylation in ileal tissues within the same time course as isometric potentiation. Both putrescine and dihydrotestosterone induced Ca²⁺ sensitization of ionomycin-permeabilized ileal smooth muscle. Finally, inhibition of the Rho kinase (ROK) pathway with the specific inhibitor Y27632 completely prevented androgen-induced potentiation. In agreement, androgens elicited ROK-induced Ser¹⁹ phosphorylation of myosin light chain 2 in ileal muscle. These data indicate that androgens potentiate ileal contractile activity by an AR-dependent nongenomic mechanism involving intracellular polyamine signaling and Ca²⁺ sensitization via ROK activation.