Phosphatidylethanolamine-ceramide-ethanolaminephosphotransferase catalyses the synthesis of ceramide-phosphoethanolamine, a sphingomyelin analogue, from ceramide and phosphorylethanolamine derived from phosphatidylethanolamine. The location of this transferase in synaptic plasma membrane vesicles from rat brain was studied in comparison with sialidase and (Na(+), K(+))ATPase. Synaptic plasma membrane vesicles were purified by sucrose gradient followed by an aqueous polymer two-phase partitioning system. Most of the vesicles were right-side out scaled as demonstrated by latency of ouabain-sensitive (Na(+), K(+))ATPase activity which was unmasked by preincubation with detergents (deoxycholate, Triton X-100 or sodium dodecyl sulphate). Detergents increased sialidase activity but transferase activity was not affected. In the absence of detergents, preincubation with proteases did not modify ATPase activity but strongly inhibited phosphatidylethanolamine-ceramide-ethanolaminephosphotransferase and sialidase activities. Yet even in the presence of detergents, a part of the active site of the transferase was not accessible to the protease action. Our results suggest that at least a part of the active centre of the transferase is situated on the external side of synaptic plasma membrane vesicles; the other part, which is inaccessible to proteases even in the presence of detergents, is embedded in the membrane interior.