The effects of incubation temperature and allosteric modulators were studied on [3H]nitrendipine binding to guinea-pig cardiac membranes. Incubation temperature only slightly affected the ability of nifedipine and verapamil derivatives to inhibit binding. By contrast, the Ca2+ channel blockers d-cis-diltiazem and fostedil (KB-944) stimulated [3H]nitrendipine binding in a temperature-dependent manner (37 degrees greater than 25 degrees greater than 4 degrees C). The stimulatory effect of fostedil could be related to a decrease (2.3-fold at 37 degrees C) in the rate of radioligand binding site dissociation, without significant effects on association kinetics. Both fostedil and d-cis-diltiazem caused a shift to the right of the concentration-inhibition curve of tiapamil, a negative allosteric modulator of [3H]nitrendipine binding. Neither compound affected the ability of nifedipine, a competitive antagonist, to inhibit radioligand binding. This selective effect of fostedil or d-cis-diltiazem may be useful for testing whether potential Ca2+ channel blockers interact in a competitive as opposed to allosteric manner with the dihydropyridine site. Varying the incubation temperature may also be useful in detecting compounds which act as positive allosteric modulators (stimulators) of dihydropyridine binding.