During the lysogenic response to infection by bacteriophage lambda, the phage-specified cII and cIII proteins provide for the coordinate establishment of repression and integration of the viral DNA. One critical regulatory function of cII/cIII is an activation of synthesis of the cI protein, the repressor that maintains lysogeny. The mechanism and site for regulation of the cI gene by cII/cIII have been a subject of controversy. The two principal hypotheses for cII/cIII action are: initiation of new RNA chains in the y region of lambda DNA just to the left of the cII gene; or antitermination of a short leader RNA (4S or oop RNA) initiated to the right of the cII gene. In an effort to distinguish between these hypotheses, we have studied the cII-mediated turn-on of cI protein synthesis in three classes of prophage deletion strains: deletions of the 4S RNA promoter but not the y region, deletions that remove both regions, and deletions that leave both intact. We find that an intact y region is required for normal regulation of the cI gene by cII, but the 4S RNA promoter is not. From experiments with other mutants, we conclude that rightward transcription from the early lytic promoter is also not necessary for positive regulation. Our results suggest that positive regulation by cII/cIII involves initiation of new RNA chains through activation of promoter sites.