Campylobacter jejuni encodes a cytolethal distending toxin (CDT) that causes cells to arrest in the G(2)/M transition phase of the cell cycle. Highly related toxins are also produced by other important bacterial pathogens. CDT activity requires the function of three genes: cdtA, cdtB, and cdtC. Recent studies have established that CdtB is the active subunit of CDT, exerting its effect as a nuclease that damages the DNA and triggers cell cycle arrest. Microinjection of CdtB into target cells led to G(2)/M arrest and cytoplasmic distention, in a manner indistinguishable from that caused by CDT treatment. Despite this progress, nothing is known about the composition of the CDT holotoxin or the function of CdtA and CdtC. We show here that, when applied individually, purified CdtA, CdtB, or CdtC does not exhibit toxic activity. In contrast, CdtA, CdtB, and CdtC when combined, interact with one another to form an active tripartite holotoxin that exhibits full cellular toxicity. CdtA has a domain that shares similarity with the B chain of ricin-related toxins. We therefore proposed that CDT is a tripartite toxin composed of CdtB as the enzymatically active subunit and of CdtA and CdtC as the heterodimeric B subunit required for the delivery of CdtB.