Purified cardiac sarcolemmal membrane vesicles were used to determine if specific prostaglandin (PG) receptors are present on the myocyte. Two binding sites for PGE2were identified in isolated bovine sarcolemmal membranes: a high-affinity site with a dissociation constant (K4) of 032 nM and a maximum binding (Bmaxof 376 fmol/mg of protein and a lower-affinity site with a K4of 3.41 nM and a Bmaxof 2,112 fmol/mg of protein. In competition experiments, unlabeled PGE1displaced3H]PGE2from its membrane receptor at concentrations similar to those of unlabeled PGE2. Both PGF2μ and PGD2displaced [3H]PGE2from the membrane, but only at high concentrations (> 10−6M and > 10−5M, respectively). Digestion of sarcolemmal membrane with trypsin resulted in a threefold decrease in specific [3H]PGE2binding. Phosphorylation of the membrane with protein kinase A also decreased specific [3HJPGE2binding. At concentrations of PGE2} that occupy the high-affinity site, sarcolemmal adenylate cyclase activity was inhibited in the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p]. We conclude that the isolated cardiac sarcolemmal membrane contains a high-affinity binding site for PGEj that is functionally coupled to adenylate cyclase. The binding site is stereospecific and probably recognizes the 9-keto, ll-hydroxyl portion of the ring structure of these prostaglandins.