The role of cerebroside sulfate in the specific binding of opiates to the synaptic membrane was investigated by the use of the enzyme, sulfatase A (cerebroside sulfatase, cerebroside-3-sulfate-3-sulphohydrolyase, EC 220.127.116.11). It was observed that the hydrolysis of sulfatides in the synaptic membrane by this enzyme was dependent on the presence of a heat stable protein molecule. When [3H]naloxone binding to the enzyme-treated membrane was determined, specific binding was attenuated. This inhibition of specific binding was dependent on the concentration of the activator molecule and could not be prevented by the addition of bovine serum albumin in the binding assay mixtures. The enzymatic pH optimum for inhibiting the specific opiate binding was between pH 4.7 and 5.0. This correlated well with the pH optimum for the sulfatase A to hydrolyze free cerebroside sulfate molecules. Scatchard analysis indicated a significant decrease in the number of binding sites with no statistically significant alteration in binding affinity of the remaining sites.