We have analyzed the kinetics of transcription initiation and reinitiation in vitro by one of the simplest and best characterized transcription machineries, bacteriophage T7 RNA polymerase (T7 RNAP). We used a short transcription unit with U-specific promoter and terminator elements as a template, and a heparin challenge assay to distinguish the first transcription cycle from the subsequent ones. When present at sub-saturating concentrations with respect to template DNA, T7 RNAP could find its promoter and initiate the first transcription cycle in less than 1 min. Reinitiation under the same conditions proceeded more slowly, with only three new transcription cycles being completed in 10 min; after that time, reinitiation practically ceased. When the polymerase was in large excess over template DNA, however, reinitiation proceeded linearly for longer times, at a rate of 1 cycle/min. Our data suggest that polymerase recycling represents a critical step in T7 RNAP transcription, and that such a step may become rate-limiting for transcription at sub-saturating polymerase concentrations. (C) 2004 Elsevier Inc. All rights reserved.