The recently discovered endomorphin 1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin 2 (Tyr-Pro-Phe-Phe-NH2) were investigated with respect to their direct receptor-binding properties, and to their ability to activate G proteins and to inhibit adenylyl cyclase in both cellular and animal models. Both tetrapeptides activated G proteins and inhibited adenylyl cyclase activity in membrane preparations from cells stably expressing the mu opioid receptor, an effect reversed by the mu receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2), but they had no influence on cells stably expressing the delta opioid receptor. To further establish the selectivity of these peptides for the mu opioid receptor, brain preparations of mice lacking the mu opioid receptor gene were used to study their binding and signalling properties. Endomorphin 2, tritiated by a dehalotritiation method resulting in a specific radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), labelled the brain membranes of wild-type mice with a Kd value of 1.77 nM and a Bmax of 63.33 fmol/mg protein. In membranes of mice lacking the mu receptor gene, no binding was observed, and both endomorphins failed to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding and to inhibit adenylyl cyclase. These data show that endomorphins are capable of activating G proteins and inhibiting adenylyl cyclase activity, and all these effects are mediated by the mu opioid receptors.