The effects of prostaglandins on the adenylate cyclase-cyclic adenosine 3',5'-monophosphate (cAMP) system of rat liver were studied both in vitro and in vivo and compared to those of glucagon and isoproterenol. Prostaglandin (PG) E,, 4 x 10−4M to 2 x 10−5M, significantly increased hepatic adenylate cyclase activity in vitro. The stimulation of adenylate cyclase by PGE, was potentiated by addition of 8 μM GTP to the incubation media. By contrast, GTP did not augment the slight increase in adenylate cyclase activity seen in response to the prostaglandin ethanol control. However, the maximum stimulation of adenylate cyclase observed in response to PGE1 (4-fold) was considerably less than that obtained with glucagon (6-fold), but similar in magnitude to the response mediated by isoproterenol. Intraportal vein administration of 40 μg of PGE, to intact rats increased hepatic cAMP content 2-fold at 3 min after injection. The in-vivo response to PGE, was also comparable to that seen with isoproterenol but much less than that observed with glucagon (20-fold increases in 15 sec). Both PGE1 and glucagon increased the cAMP content in rat liver perfused in situ, further supporting a direct action of PGE1 on the hepatic adenylate cyclasecAMP system. PGE, had no detectable effects on hepatic cyclic phosphodiesterase activity in vitro. Therefore, the observed increases in cAMP mediated by PGE, were probably due to an increase in cAMP synthesis. In contrast to PGE1, PGF2α and PGA, did not significantly alter the rat hepatic adenylate cyclasecAMP system in vitro or in vivo, while PGE2 had a modest stimulatory action. The in vitro stimulation of hepatic adenylate cyclase by PGE2, like that of PGE1, was augmented by the addition of GTP, whereas the latter did not alter the action of PGF2α. (Endocrinology94: 1404, 1974)