The androgenic steroid hormone testosterone induced an early (<30–60 seconds) stimulation of endocytosis, hexose transport, and amino acid transport, monitored by the temperature-sensitive uptake of horseradish peroxidase, 2-deoxyglucose, and α-aminoisobutyrate, respectively, in rat ventricle cubes and acutely isolated ventricular myocytes. This stimulation was time-and concentration-dependent and was maximal at 10-9to 10-8M testosterone, consistent with androgen-receptor mediation. EGTA (2.5 mM), La3+(1 mM), and verapamil (100 μM) ablated the hormonal response. The calcium ionophore A23187 (10 μM) induced an acute stimulation of endocytosis, amino add transport, and hexose transport which was not further increased by testosterone (10-8M), suggesting a common effector pathway. Testosterone (10-8M) also evoked a rapid (<30 seconds) stimulation of45Ca influx and efflux. Testosterone (10-8M) induced a rapid (<5 seconds) transient increase in ornithine decarboxylase (ODC) activity peaking (twofold to threefold) at 60 seconds, and an early (15 seconds) transient accumulation of polyamines peaking at 60 seconds In isolated myocytes. The specific, irreversible ODC inhibitor α-difluoromethylornithine (DFMO, 5-10 mM) blocked the testosterone-evoked increase in ODC activity and polyamine levels and the stimulation of Ca2+fluxes, endocytosis, hexose transport, and amino acid transport. Putrescine (0.5-1 mM), the ODC product, reversed DFMO inhibition and restored the increase in polyamines,45Ca fluxes, and Ca2+-dependent membrane transport processes. These results demonstrate that rapid, transient ODC-regulated polyamine synthesis is essential for androgenic stimulation of Ca2+fluxes and membrane transport processes in ventricular myocytes. These findings support a model for signal transduction in which newly synthesized polyamines serve as intracellular messengers to regulate transmembrane Ca2+movements, Ca2+-dependent membrane transport functions, and other Ca2+-and polyamine-sensitive processes in cardiac myocytes.