The binding of the 1,4-dihydropyridine calcium channel blocker [3H]nitrendipine to canine cardiac sarcolemmal and bovine aortic membranes was found to be rapid, specific, saturable, and reversible. Dissociation constants (Kd) determined by Scatchard analysis were 0.14 and 0.16 nM and the maximal numbers of binding sites (Bmax) were 0.96 +/- 0.2 and 0.08 +/- 0.01 pmole/mg protein for cardiac and aortic membranes respectively. Values of Kd calculated from kinetic data were approximately 0.10 nM for both membrane preparations. Competition assays with the enantiomers of a nisoldipine derivative indicated that [3H]nitrendipine binds stereoselectively. The order of potency of several nifedipine analogs for inhibition of binding of [3H]nitrendipine to cardiac and aortic membranes paralleled their relative potencies for inhibition of contraction in smooth muscle. It is concluded that the high affinity binding sites for nitrendipine in bovine aortic smooth muscle membranes are similar to those of canine ventricular sarcolemma.