The dihydropyridine receptor is associated with the L-type Ca 2+ channel in the cell membrane. In this study we have examined the effects of group-specific modification on dihydropyridine binding in heart sarcolemmal membranes isolated from the rabbit. Specifically, dithiothreitol and glutathione were employed to assess the possible role of disulfide (-SS-) bonds in the binding of [ 3H]dihydropyridines. NEM, PCMS and iodoacetamide were employed to examine the effect of blocking free sulfhydryl groups (-SH) on the binding of [ 3H]dihydropyridines to their receptor in heart sarcolemma. Glutathione inhibited [ 3H]PN200-110 binding to sarcolemmal membranes 100%, with an IC 50 value of 50 μM, while DTT inhibited maximally by 75% with an IC 50 value in the millimolar range. Alkylation of free sulfhydryl groups by NEM or iodoacetamide inhibited binding of [ 3H]PN200-110 binding in cardiac sarcolemma approx. 40–60%. Blocking of free sulfhydryl groups by PCMS completely inhibited [ 3H]PN200-110 binding to their receptor in sarcolemmal membranes in a dose-dependent manner with an IC 50 value of 20 μM. These results suggest the involvement of disulfide bonds and free sulfhydryl groups in DHP binding to the L-type Ca 2+ channel in heart muscle. We also examined the effect of membrane phosphorylation on the specific binding of the dihydropyridine [ 3H]nitrendipine to its receptor. Phosphorylation was studied in cardiac sarcolemmal as well as skeletal muscle transverse-tubule membranes. Phosphorylation due to endogenous protein kinase and cAMP-dependent protein kinase was without effect on [ 3H]nitrendipine binding in both cardiac sarcolemmal and skeletal muscle membranes. Addition of exogenous calmodulin under conditions known to promote Ca 2+/calmodulin-dependent phosphorylation increased [ 3H]nitrendipine binding 20% with no alteration in K D in both types of membrane preparation. These results suggest a role for calmodylin in dihydropyridine binding to L-type Ca 2+ channels.