Sphingosine inhibits the activity of the skeletal muscle Ca2+ release channel (ryanodine receptor) and is a noncompetitive inhibitor of [3H]ryanodine binding (Needleman et al., Am. J. Physiol. 272, C1465-1474, 1997). To determine the contribution of other sphingolipids to the regulation of ryanodine receptor activity, several sphingolipid bases were assessed for their ability to alter [3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes and to modulate the activity of the Ca2+ release channel. Three lipids, N,N-dimethylsphingosine, dihydrosphingosine, and phytosphingosine, inhibited [3H]ryanodine binding to both skeletal and cardiac SR membranes. However, the potency of these three lipids and sphingosine was lower in rabbit cardiac membranes when compared to rabbit skeletal muscle membranes and when compared to sphingosine. Like sphingosine, the lipids inhibited [3H]ryanodine binding by greatly increasing the rate of dissociation of bound [3H]ryanodine from SR membranes, indicating that these three sphingolipid bases were noncompetitive inhibitors of [3H]ryanodine binding. These bases also decreased the activity of the Ca2+ release channel incorporated into planar lipid bilayers by stabilizing a long closed state. Sphingosine-1-PO4 and C6 to C18 ceramides of sphingosine had no significant effect on [3H]ryanodine binding to cardiac or skeletal muscle SR membranes. Saturation of the double bond at positions 4-5 decreased the ability of the sphingolipid bases to inhibit [3H]ryanodine binding 2-3 fold compared to sphingosine. In summary, our data indicate that other endogenous sphingolipid bases are capable of modulating the activity of the Ca2+ release channel and as a class possess a common mechanism of inhibition.