Extracts prepared from HeLa cell nuclei are inactivated for accurate transcription from the adenovirus 2 major late promoter (Ad2 MLP) by repeated passage over a column containing immobilized calf thymus RNA polymerase II. The capacity for accurate transcription is restored to these extracts by a fraction that is eluted from the RNA polymerase II column with 0.5 M KC1. This fraction contains RNA polymerase II-associating proteins (RAPs). RAPs are not required for the formation of a stable, promoter-associated pre-initiation complex. However, at least one RAP is required for the formation of the first phosphodiester bond at the Ad2 MLP, both when initiation depends only on the TATAAA box, and when initiation is stimulated by the upstream sequence of the promoter. Initiation occurs approximately 1 min after the addition of RAPs to initiation complexes formed in the absence of RAPs. Therefore, the RAP(s) that is an RNA polymerase II initiation factor functions late in the initiation pathway to convert a stable preinitiation complex to an initiation-competent complex.