To determine whether calcium channel agonists and antagonists bind to distinct pharmacologically active sites, the binding of dihydropyridine calcium channel agonists and antagonists was related to calcium flux and contractile state in primary monolayer cultures of spontaneously contracting chick embryo ventricular cells. Equilibrium binding studies using the antagonist (+)-[3H]PN200–110 demonstrated equilibrium binding to intact, beating cells consistent with a single class of binding sites (KD, 1.1 nM; Bmax, 40 fmol/mg protein). Membrane depolarization of the intact cells by incubation in 30 mM potassium caused a 91 % increase in the apparent number of (+)-PN200–110 binding sites (Bmax76 fmol/mg protein), but no significant change in the KD(1.2 nM). The (+)-PN200–110 produced a concentration-dependent decrease in calcium uptake (IC502.2 nM) and contractile amplitude (IC505.6 nM). The calcium channel agonist, (±)-[3H]BAY k 8644, bound to two distinct binding sites with high affinity (KD1.0 nM) and low affinity (KD1.9 μM). The (±)-BAY k 8644 produced biphasic modulation of calcium flux and contractile state. At concentrations of 100 nM or less, (±)-BAY k 8644 increased calcium flux and contractile amplitude, consistent with drug interaction with the high affinity agonist site. However, at higher concentrations, the stimulatory effect of (±)-BAY k 8644 on calcium flux and contractile amplitude was abolished, a finding that is consistent with drug interaction with the low affinity antagonist site. Ligand binding studies using (±)-BAY k 8644 to displace (+)-[3H]PN200–110 demonstrated that these ligands compete at a single site with KDfor (±)-Bay k 8644 of 1.7 μM, which is consistent with binding at the low-affinity antagonist site. It was concluded that in intact cultured myocytes, voltage-sensitive calcium channels have at least 2 distinct dihydropyridine binding sites. Binding of dihydropyridines to these sites mediates opposing effects on calcium flux and contractile function.