In order to determine the opioid receptor binding in the rat cardiac sarcolemma, direct and displacement binding assays of tritiated bremazocine, a nonselective opioid, agonist and U69593, a specific kappa-receptor agonist, were performed in a sarcolemmal membrane preparation. Saturation studies revealed that there were substantial binding sites for both radioligands. The [3H]bremazocine had a binding capacity of 100 fmol/mg protein with a Kd of 3.4 nM while the binding of [3H]U69593 showed a Kd of 7.4 nM and a Bmax of 139 fmol/mg protein. Displacement binding assay showed that [D-Ala2, MePhe4, Gly-(ol)5]enkephalin (DAGO), a specific mu-agonist, exhibited no inhibitory effect on the binding of either [3H]bremazocine or [3H]U69593. [D-Ala2,D-Leu5]enkephalin (DADLE), a specific delta-agonist, had no inhibition on the binding of [3H]U69593, but displaced the binding of [3H]bremazocine at higher concentrations (10(-5)-10(-6)M). U50488, a specific kappa-agonist, completed with the binding of [3H]U69593 23 times more potently than with the binding of [3H]bremazocine. A residual binding sites of [3H]bremazocine was found with a Kd of 6.4 nM and a Bmax of 76 fmol/mg protein under conditions in which mu, delta and kappa 1 opioid bindings were suppressed by DAGO and DADLE at 200 nM and U50488 at 1 microM. DADLE at 5 microM, a concentration known to block kappa 2 binding sites, abolished the residual U50488-insensitive specific binding of [3H]bremazocine. Met5-Enkephalin-Arg6-Phe7, a specific kappa 2 ligand, displaced the binding of [3H]bremazocine much more readily than the binding of [3H]U69593. The present study provided evidence for the presence of two kappa-receptor subtypes in the cardiac sarcolemma: one U50488-sensitive and DADLE-insensitive, and the other U50488-insensitive and DADLE-sensitive. These two kappa-receptor binding subtypes may correspond to kappa 1 and kappa 2 receptor subtypes. Computer assisted non-linear regression analysis in the competition experiments of U50488 with [3H]U69593 afforded a best fit of the inhibition curves for two displacing components of kappa 1 binding sites: one with high affinity and low Bmax, another with low affinity and high Bmax.