1. In a sarcoplasmic reticulum fraction obtained from rat hearts, the analysis of equilibrium [3H]-ryanodine binding showed high and low affinity sites (KD = 1.3 nM and 2.8 microM, Bmax = 2.2 pmol mg-1 and 27.8 pmol mg-1). The dissociation rate constant increased at 1 microM vs 4 nM [3H]-ryanodine concentration, and micromolar ryanodine slowed the dissociation of nanomolar ryanodine. 2. The binding of 4 nM [3H]-ryanodine was not affected by gallopamil, while the binding of 100 nM to 18 microM [3H]-ryanodine was partly displaced. Data analysis suggested that gallopamil inhibited low affinity [3H]-ryanodine binding, with IC50 in the micromolar range. 3. Gallopamil decreased the dissociation rate constant of 1 microM [3H]-ryanodine. While gallopamil alone did not affect the dissociation of 4 nM [3H]-ryanodine, gallopamil and micromolar ryanodine slowed it to a greater extent than micromolar ryanodine alone. 4. Our results are consistent with the hypothesis that the ryanodine receptor is a negatively cooperative oligomer, which undergoes a sequential alteration after ryanodine binding. Gallopamil has complex actions: it inhibits ryanodine binding to its low affinity site(s), and probably modulates the cooperativity of ryanodine binding and/or the transition to a receptor state characterized by slow ryanodine dissociation. These molecular actions could account for the previously reported effect of gallopamil on the sarcoplasmic reticulum calcium release channel.