Shigella cytotoxin was purified from the culture medium of Shigella shigae 60 R and from the bacterial pellet by an extensive purification scheme involving chromatography on Cibachron Blue F3GA, chitin, and DEAE-cellulose columns, sucrose gradient centrifugation, and finally polyacrylamide gel electrophoresis under nondenaturing conditions. In sodium dodecyl sulfate polyacrylamide gels, purified toxin showed two bands, a heavy one with the molecular weight of 30,500 (A chain), and a broad band migrating near the dye front (B chain). The A chain was easily cleaved by proteases into two fragments, A1 (Mr = 27,500) and A2 (Mr = 3,000). These fragments were linked with a disulfide bridge (nicked toxin). The molecular weight of native toxin was estimated by the method of Ferguson to be 68,000 +/- 4,000. Cross-linking experiments indicated that the native toxin consists of one A chain and six to seven B chains, with each B chain having a molecular weight of congruent to 5,000. The isolated A and B chains were not toxic to cells nor did they bind to cells to a measurable extent. In a cell-free system, the A1 fragment strongly inhibited protein synthesis. The possibility that the B chains form the binding moiety of the toxin is discussed.