The CIII protein of lambdoid bacteriophages promotes lysogeny by stabilizing the phage-encoded CII protein, a transcriptional activator of the repressor and integrase genes. We have isolated a set of missense mutations in the cIII gene of phage lambda and of phage HK022 that yield inactive CIII proteins. All the mutations are located in the relatively conserved central region of the protein. A comparative analysis of the CIII protein sequence in lambda, HK022, and the lambdoid bacteriophage P22 leads us to suggest that this central region assumes an amphipathic alpha-helical structure. This part of the lambda cIII gene was cloned within a fragment of the lacZ gene (the alpha-complementing fragment). The resulting fusion protein displays CIII activity. Mutations that yield a nonfunctional fusion protein map within its CIII moiety. These results indicate that the central portion of the CIII protein is both necessary and sufficient for CIII activity.