Cyclic AMP produced from membrane receptor complex bound adenylyl cyclases is protective in corneal endothelial cells (CEC). CEC also express soluble adenylyl cyclase (sAC), which is localized throughout the cytoplasm. When activated by HCO(3)(-), cAMP concentration ([cAMP]) increases by ∼50%. Here we ask if cAMP produced from sAC is also protective. We examined the effects of HCO(3)(-), pH, phosphodiesterase 4 inhibition by rolipram, sAC inhibition by 2HE (2-hydroxyestradiol), and sAC small interfering RNA (siRNA) knockdown on basal and staurosporine-mediated apoptosis. HCO(3)(-) (40 mM) or 50 μM rolipram raised [cAMP] to similar levels and protected endothelial cells by 50% relative to a HCO(3)(-)-free control, whereas 2HE, which decreased [cAMP] by 40%, and H89 (PKA inhibitor) doubled the apoptotic rate. sAC expression was reduced by two-thirds in the absence of HCO(3)(-) and was reduced to 15% of control by sAC siRNA. Protection by HCO(3)(-) was eliminated in siRNA-treated cells. Similarly, caspase-3 activity and cytochrome c release were reduced by HCO(3)(-) and enhanced by 2HE or siRNA. Analysis of percent annexin V+ cells as a function of [cAMP] revealed an inverse, nonlinear relation, suggesting a protective threshold [cAMP] of 10 pmol/mg protein. Relative levels of phosphorylated cAMP response element binding protein and phosphorylated Bcl-2 were decreased in CEC treated with 2HE or siRNA, suggesting that HCO(3)(-)-dependent endogenous sAC activity can mobilize antiapoptotic signal transduction. Overall, our data suggest a new role for sAC in endogenous cellular protection.