While many observations indicate that prostaglandins may act as positive regulators of hepatocyte proliferation, the underlying mechanisms are not known. We have examined some of the signal pathways in the growth response induced by prostaglandins in hepatocytes, with particular focus on adenylyl cyclase and phosphoinositide-specific phospholipase C. Adult rat hepatocytes were cultured as primary monolayers in serum-free medium in the presence of EGF and insulin. PGE2 or PGF2 alpha (added 0-3 h after plating) enhanced the incorporation of [3H]-thymidine into DNA (measured at 50 h); at 100 microM the stimulation was about threefold PGI2 and PGD2 also showed significant but smaller stimulatory effects. No significant increase in the level of cyclic AMP (cAMP) was detected in response to any of the prostaglandins. Low concentrations of glucagon (0.1-10 nM), a potent activator of hepatic adenylyl cyclase, or 8-bromo-cAMP (0.1-10 microM) enhanced the DNA synthesis. When 8-bromo-cAMP was used in maximally effective concentrations, no further stimulation was obtained by combining it with glucagon, whereas the effects of PGE2 and 8-bromo-cAMP were completely additive. All the prostaglandins also showed additivity with the effect of glucagon on the DNA synthesis. PGE2, PGF2 alpha, PGI2, and PGD2 increased intracellular inositol-1,4,5-trisphosphate (InsP3), with a relative order of efficacy roughly corresponding to their activity as stimulators of DNA synthesis. Increases in cytosolic free Ca2+, as measured in single cells, were elicited in a majority of the hepatocytes by all these prostaglandins at 1 microM. Supramaximal concentrations of vasopressin, a strong activator of phospholipase C in hepatocytes, acted additively with PGE2 on the DNA synthesis. Pretreatment of the hepatocytes with a concentration of pertussis toxin that prevented the inhibitory effect of PGE2 on glucagon-induced cAMP accumulation did not abolish the ability of PGE2 to stimulate the DNA synthesis. The results do not support a role for adenylyl cyclase activation in the stimulatory effect of prostaglandins on hepatocyte growth. While the data are compatible with an involvement of phosphoinositide-specific phospholipase C in the growth-promoting effect of prostaglandins in cultured rat hepatocytes, they suggest this may not be the sole mechanism.