Insertion of DNA containing PR, the early rightward promoter of bacteriophage lambda, is lethal to M13-derived vectors when the promoter directs transcription (using the '+' strand as template) toward the M13 origin of replication (ori). Lethality can be relieved by mutation of PR, repression of the promoter by the lambda cl repressor, or by insertion of a strong transcription terminator between PR and ori. We have used selection for plaque formation in the absence of repressor to isolate 14 different mutations at 8 sites in PR. This method of isolating promoter mutants in vivo is applicable generally to strong promoters whose activity is regulated either positively or negatively.