Diphtheria toxin contains a trypsin-sensitive region with 3 closely spaced arginines in the sequence (Asn189, Arg190, Val191, Arg192, Arg193, Ser194). Cleavage of the toxin to yield A- and B-fragments ("nicking") appears to occur in a stochastic manner after either of these arginine residues. Isoelectric focusing of A-fragment prepared in vitro showed four bands of varying intensity with pI between 4.5 and 5.0, three of which could be accounted for by the three different cleavage sites. Exposure of cells with surface-bound toxin to pH less than 5.3 induces translocation of A-fragment to a position where it is shielded from external Pronase, presumably in the cytosol. A-fragment translocated in this manner had the same pI as the most acidic A-fragments, indicating that only A-fragments lacking both Arg192 and Arg193 are translocation-competent. This was confirmed by amino acid sequencing. Treatment of A-fragment with carboxypeptidase B eliminated the two bands with the highest pI while there was a concomitant increase in the bands corresponding to the two most acidic A-fragments. Such treatment of nicked diphtheria toxin increased the amount of translocated A-fragment and the ability of toxin to form cation-selective pores in the cell membrane. The site of trypsin cleavage therefore appears to be one of the factors limiting toxin entry to the cytosol.