The binding of [3H]BAY K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)- pyridine-5-carboxylate] to high and low affinity sites in rabbit ventricular membranes was characterized. Binding affinities were 0.66 and 138 nM at 15 degrees C and 9.1 and 72 nM at 37 degrees C, for the high and low affinity sites, respectively, and binding site densities were 0.3 and 14 pmol/mg at 15 degrees C and 0.41 and 1.4 pmol/mg at 37 degrees C, for the respective sites. The modification of high affinity [3H]BAY K 8644 binding by verapamil, diltiazem, tiapamil, Ca++ and EDTA appeared to be the same as that for nitrendipine binding, consistent with the hypothesis that the high affinity binding site for [3H]BAY K 8644 on isolated membranes is the same as the 1,4-dihydropyridine antagonist binding site. The binding of [3H]BAY K 8644 to a low affinity binding site was modified by temperature, Ca++ and diltiazem, but the lack of stereoselectivity, lack of denaturation by heat and the large number of sites indicated that most of the low affinity binding sites were not associated with Ca++ channels. It is concluded that the high affinity binding site for BAY K 8644 is associated with Ca++ channels, and is modified by at least some of the factors that modify the binding site for Ca++ channel antagonists, whereas many or all of the low affinity binding sites detected are not related to Ca++ channels.