The mutation, prmE37, located at —14 in the P RM promoter of bacteriophage λ, reduces P RM function dramatically both in vitro and in vivo. In a search for second-site revertants of prmE37, we isolated a double mutant that exhibits a partially restored Prm + phenotype. The second-site mutation (at —31) is identical to the mutation prmup-1. The activity of the doubly mutant (pseudo-revertant) promoter, prmE37 prmup-1, was investigated in vivo using a P RM- lacZ fusion phage and found to be intermediate between that of prmE37 and wild-type P RM. However, the relative strength of the prmE37 prmup-1 promoter was greater than expected following superinfection of a λ lysogen. Since nalidixic acid was found to preferentially inhibit transcription from the doubly mutant promoter under these conditions, we suggest that DNA supercoiling favors activation of this promoter by represser. In runoff transcription assays in the absence of repressor, the activity of wild-type P RM and the doubly mutant promoter were the same. However, while addition of repressor significantly stimulated wild-type P RM, it had little or no effect on the activity of the doubly mutant promoter. Values of K B, the equilibrium constant for formation of closed complexes, and k f, the rate constant for isomerization of closed to open complexes, were determined in abortive initiation assays, and the product k f K B was used as a measure of promoter strength. The results of these assays are in agreement with those obtained in runoff transcription assays. In the absence of repressor, values of k f K B for the doubly mutant promoter and wild-type P RM are the same; however, τ obs, the time required for open complex formation, is significantly greater for the double mutant than for wild-type P RM at all RNA polymerase concentrations used for the abortive initiation analysis. In the presence of repressor, the doubly mutant promoter is stronger than the prmE37 promoter, but much weaker than wild-type P RM. This is due to the fact that k f for the doubly mutant promoter is increased 2.5-fold by repressor, but K B is reduced to the same extent. These two effects counteract each other, so that repressor has no net effect on the strength of the prmE37 prmup-1 promoter in vitro. In contrast, repressor increases k f for wild-type P RM eightfold and increases overall promoter strength ( K B K f) nearly fivefold. In the presence of repressor, the effects of the two mutations, prm E37 and prm up-1, on k f are independent. This observation is discussed in relation to revised models for open complex formation.