When the transcription startsites of the phage lambda promoters PRM and PR are separated by 82 bp (the wild-type spacing), mutating PR increases the rate of open complex formation at PRM at all RNA polymerase (RNAP) concentrations tested in vitro. This is reflected in a fourfold increase in kappa f (the rate constant for isomerization of closed to open complexes) and a threefold decrease in KB (the equilibrium constant for formation of closed complexes). These effects of mutating PR resemble qualitatively those we observed when the separation between the two promoters was decreased by a single base-pair, but are quantitatively less dramatic. Although mutating PR has the additional effect of uncovering a weak promoter, P alpha, which overlaps both PRM and PR, the presence of P alpha does not account for the effects of PR mutations on open complex formation at PRM. In fixed-time assays at a single RNAP concentration, repressor stimulated PRM approximately threefold on a PR- template, indicating that activation is mediated substantially by a direct interaction between repressor and RNAP. That is, activation of PRM is not merely an indirect consequence of repressing PR. Kinetic data confirm this conclusion. In a PR- genetic background, repressor increased kappa f six- to eightfold and decreased KB approximately twofold. Similar results were obtained when OR3 was mutated, indicating that the effect on KB is not due to repressor binding to OR3. Thus, repressor causes a significant increase in the rate of open complex formation at PRM even when PR is inactive. On a PR+ template, 75 nM repressor stimulated PRM by increasing kappa f eightfold, with no effect on KB, which agrees with previous results. However, increased repressor concentrations stimulated kappa f by an additional factor of two to four, indicating that previous experiments underestimated the effect of repressor on kappa f. At the same time, increasing the repressor concentration decreased KB for PRM on a wild-type template. At the highest repressor concentration tested (275 nM), KB decreased 15-fold, presumably due to OR3-mediated repression of PRM. However, at an intermediate repressor concentration (170 nM) values of kappa f and KB for PRM on a PR+ template were in close agreement with the corresponding parameters obtained on a PR- template. These data lead us to suggest that repressor causes a decrease in KB for PRM on both a PR+ and a PR- template independent of its ability to bind to OR3.