Cell-associated enterotoxin B was detected in lysates of cells of Staphylococcus aureus S-6 and 4916 disrupted by sonication or lysostaphin treatment. As much as 67% of this total cell-associated toxin was surface-bound, located outside the cytoplasmic membrane, and was released during protoplasting of this organism by lysostaphin treatment in hypertonic medium. The remainder of the cell-associated toxin was termed cytoplasmic and was released during osmotic lysis of the protoplasts. Levels of cell-associated toxin as a function of the age of the cells showed a rapid increase in both surface-bound, cytoplasmic, and total cell-associated toxin levels during the period of active toxin synthesis (late exponential phase of growth). These cell-associated toxin levels then reached a peak as the culture entered stationary phase, at a time corresponding to a decrease in the rate of toxin synthesis, and decreased slowly thereafter.