The formation of the transcriptionally competent open complex (RP(o)) by Escherichia coli RNA polymerase at the lambda P(R) promoter involves at least three steps and two kinetically significant intermediates (I(1) and I(2)). Understanding the sequence of conformational changes (rearrangements in the jaws of RNA polymerase, DNA opening) that occur in the conversion of I(1) to RP(o) requires: (1) dissecting the rate constant k(d) for the dissociation of RP(o) into contributions from individual steps and (2) isolating and characterizing I(2). To deconvolute k(d), we develop experiments involving rapid upshifts to elevated concentrations of RP(o)-destabilizing solutes ("perturbants": urea and KCl) to create a burst in the population of I(2). At high concentrations of either perturbant, k(d) approaches the same [perturbant]-independent value, interpreted as the elementary rate constant k(-2) for I(2)-->I(1). The large effects of [urea] and [salt] on K(3) (the equilibrium constant for I(2) is in equilibrium with RP(o)) indicate that a large-scale folding transition in polymerase occurs and a new interface with the DNA forms late in the mechanism. We deduce that I(2) at the lambda P(R) promoter is always unstable relative to RP(o), even at 0 degrees C, explaining previous difficulties in detecting it by using temperature downshifts. The division of the large positive enthalpy change between the late steps of the mechanism suggests that an additional unstable intermediate (I(3)) may exist between I(2) and RP(o).