Expression of the gene of the methanol-soluble, heat-stable enterotoxin of Escherichia coli (STA) allowed the identification by SDS-PAGE of a cell-associated 7500 Dalton STA-related peptide; when similar experiments were performed with a phosphate buffer SDS-PAGE system, an additional Mr 9800 band became apparent. The 9800 Dalton form, pre-pro-STA, accumulated as an intracellular species when the experiments were performed in the presence of the proton ionophore CCCP (carbonylcyanide m-chlorophenylhydrazone); by pulse-chase experiments, it was shown that pre-pro-STA became a periplasmic Mr 7500 pro-STA and this form was chased to the culture supernatant; periplasmic and extracellular pro-STA showed the same electrophoretic mobility. A short time after the pulse, pro-STA was converted extracellularly to mature STA (Mr 4500). It is proposed that STA is synthesized as pre-pro-STA, a 72-amino-acid peptide that is subsequently cleaved between amino acids 19 and 20 as it is translocated across the inner membrane. The resulting 53-amino-acid pro-STA is first detected in the periplasm and is then secreted to the culture supernatant. Pro-STA is cleaved extracellularly to yield mature STA (Mr 4500).