We have previously demonstrated that decreased O₂ tension inhibits prostaglandin synthesis from human corpus cavernosum smooth muscle cells in static culture over 8-18 h (R. B. Moreland et al., Molecular Urology 2: 41-47, 1998). In this report, an experimental system was designed that allowed determination of the effects of O₂ tension changes over the time frame of physiological penile erection. Human corpus cavernosum smooth muscle cells were cultured on microcarrier beads in enclosed stirrer flasks so that rapid changes of O₂ tension could be modulated. After 18 h of equilibration at 30-40 mmHg to simulate blood PO₂ at penile flaccidity, O₂ tension was increased to 100 mmHg for 1 h and then returned to 30-40 mmHg. Media samples were withdrawn for prostanoid synthesis and cell samples were taken for cAMP determinations. After 18 h of 30-40 mmHg PO₂ values, prostanoid synthesis by human corpus cavernosum smooth muscle cells was low (0.1-0.7 pmol/10⁶ cells). When PO₂ was increased to 100 mmHg, a rapid increase in PGE₂ >> PGF₂ > PGD₂ was observed (thromboxane A₂ was undetectable), which peaked at 5.7 pmol PGE₂/10⁶ cells. Increased O₂ tension correlated with increased PGE₂ and increased intracellular synthesis of cAMP. The prostaglandin G/H synthase inhibitor indomethacin or the E prostanoid (EP₂)-selective antagonist AH-6809 each inhibited the O₂-tension-dependent increases in cAMP. These data support a role of differential O₂ tension in the penis in the smooth muscle synthesis of PGE₂, which in turn increases cAMP synthesis via EP₂ receptors.