An extract capable of selectively transcribing early vaccinia virus genes was prepared by disrupting purified vaccinia virions and passing the soluble material through a DEAE-cellulose column to remove endogenous DNA. Runoff transcripts of predicted size were synthesized by using double-stranded DNA templates that contained truncated early vaccinia virus genes, whereas several late vaccinia virus genes were not transcribed under these conditions. Proper dilution of the enzyme extract was critical, and a threshold concentration of DNA was required. At 30 degrees C, runoff transcripts were detected after 5 min and synthesis slowed appreciably after 30 min. Mg2+ was the preferred divalent cation, and KCl concentrations above 20 mM were inhibitory. Correct initiation of transcription was demonstrated by high-resolution analysis of S1 nuclease-digested hybrids formed by annealing in vitro-synthesized RNA with 5'-end-labeled DNA. A requirement for a 31-base-pair transcriptional regulatory sequence was found by using templates with deletions in an early promoter region. This in vitro system may be useful for mapping early transcriptional initiation sites, measuring the effects of additional promoter mutations, and isolating transcription factors.