The minimum sequence of the enzymatic (A) subunit of Shiga toxin (STX) required for activity was investigated by introducing N-terminal and C-terminal deletions in the molecule. Enzymatic activity was assessed by using an in vitro translation system. A 253-amino-acid STX A polypeptide, which is recognized as the enzymatically active portion of the 293-amino-acid A subunit, expressed less than wild-type levels of activity. In addition, alteration of the proposed nicking site between Ala-253 and Ser-254 by site-directed mutagenesis apparently prevented proteolytic processing but had no effect on the enzymatic activity of the molecule. Therefore, deletion analysis was used to identify amino acid residue 271 as the C terminus of the enzymatically active portion of the STX A subunit. STX A polypeptides with N-terminal and C-terminal deletions were released into the periplasmic space of Escherichia coli by fusion to the signal peptide and the first 22 amino acids of Shiga-like toxin type II, a member of the STX family. Although these fusion proteins expressed less than wild-type levels of enzymatic activity, they confirmed the previous finding that Tyr-77 is an active-site residue. Therefore, the minimum domain of the A polypeptide which was required for the expression of enzymatic activity was defined as StxA residues 75 to 268.