Drosophila genomic DNAs containing a chromosomal locus 87C1 70,000-dalton heat shock protein gene, the locus 79B actin gene, and the 88F actin gene have been used as templates in an in vitro HeLa transcription system. RNA polymerase II-dependent transcription initiates from specific sites on the heat shock protein gene and the 79B actin gene. The locations of the transcription start sites were determined by two types of experiments: sizing of RNA runoff transcripts and S1 nuclease mapping of the 5' terminus of the in vitro transcripts. Transcription initiates at or near the in vivo initiation site of the heat shock protein gene and initiates at or near a site 14 nucleotides downstream of the in vivo start site of the 79B actin gene. The addition of the 79B actin, 88F actin, or heat shock protein templates to a HeLa extract transcription reaction precluded the transcription of a second template subsequently added. The exclusion occurs rapidly, within 15 s, is not dependent on transcription, and is only partially resistant to high concentrations of the second added template. We propose that stable protein-promoter complexes play an important role in maintaining exclusive transcription of the first template added in vitro.