Transcription initiation by RNA polymerase II from the adenovirus major late promoter was studied in vitro in a nuclear extract derived from HeLa cells. We found that different concentrations of Sarkosyl blocked transcription initiation at several different functional steps. These corresponded to two steps defined previously in a partially purified transcription system: formation of the rapid start complex, which required incubation of template with the extract and was blocked by Sarkosyl concentrations greater than 0.025%, and productive initiation, which occurred upon addition of nucleotides and was prevented by Sarkosyl concentrations greater than 0.1%. We used assays based on these findings to probe further the events that comprise the processes of initiation and reinitiation of transcription from the major late promoter. We found that the templates used in the first round of initiations from preformed rapid start complexes were more actively transcribed in the second round of initiations than newly added templates. We argued that the most likely explanation for this observation was that some transcription component(s) remained committed to the promoter after formation of an elongation complex. This template-committed complex must have been distinct from the rapid start complex, because reinitiation and rapid start complex formation were both blocked by 0.025% Sarkosyl.