The physiological conditions for the excretion of staphylococcal enterotoxin B (SEB) by nongrowing cells have been determined. In general, SEB excretion appears to be best when amino acids serve as sources of carbon, nitrogen, and energy. Little toxin was produced in a nitrogen-free medium with glucose as the sole carbon source. Inhibitor studies have shown that most of the toxin excreted by nongrowing cells was the result of de novo protein synthesis. Optimum toxin excretion was also obtained under conditions of aeration, and at or near optimum growth temperature, those conditions at which the cells are metabolically and biosynthetically most active. Levels of cell-associated SEB decreased during toxin excretion. However, these levels of toxin could not account for the high levels of toxin released during excretion. Glucose and several other rapidly fermentable carbohydrates inhibited toxin excretion by as much as 80% under conditions of uncontrolled decrease in pH: however, when the pH was kept at either 6.2 or 7.5 a stimulation of toxin excretion was observed. Protoplast studies have suggested that SEB production does not take place in the absence of an intact cell wall.