Transcription initiation by RNA polymerase II is a complex, multistep process that minimally involves transcription complex assembly, open complex formation, and promoter clearance. Hydrolysis of the beta--gamma phosphoanhydride bond of ATP has previously been shown to be required for open complex formation, as well as for the phosphorylation of the carboxy-terminal domain of the largest subunit of RNA polymerase II. The observation that ATP-dependent activation of transcription complexes can be blocked by ATP analogues that contain nonhydrolyzable beta--gamma phosphoanhydride bonds (such as ATPgammaS) was exploited to develop a functional kinetic assay for ATP-activated transcription complexes. Activated complexes on the promoter present in the long terminal repeat of the proviral DNA of mouse mammary tumor virus were defined as those that could productively initiate transcription in the presence of excess ATPgammaS. Activation is dependent on treatment of assembled preinitiation complexes with ATP (or dATP) prior to addition of ATPgammaS. At least 15-35% of the total number of preinitiation complexes present become activated within 2 min in the presence of (d)ATP, and activation appears to be rapidly reversible. The time course of formation of activated complexes in the presence of dATP is characterized by two kinetic phases: a rapid formation followed by a relatively slow decay. Activated complexes were estimated to form with a half-time of less than 1 min.