A putative precursor to staphylococcal enterotoxin B (SEB) has been identified as a component of purified membranes from Staphylococcus aureus S6. Agarose gel immunodiffusion analysis of the solubilized membranes demonstrated an immunoreactive protein that formed complete lines of identity with purified extracellular SEB. This putative precursor (pSEB) also had a different electrophoretic mobility from that of extracellular SEB when analyzed by immunoelectrophoresis. When membrane proteins from S6 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose sheets and probed with I-125 labeled, affinity-purified anti-SEB, the pSEB band was identified. The pSEB was approximately 3,500 daltons larger than extracellular SEB. This component was purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two-dimensional peptide maps of the putative SEB precursor revealed that most of the tryptic peptides were identical to those of mature extracellular SEB. When purified membranes of other SEB+ (DU4916 and 10-275) and SEB- (RN450, RN451, S6R, and FR1100) S. aureus strains were analyzed by the nitrocellulose blot procedure, only the SEB+ strains contained this putative SEB precursor on their membranes.