The transcription of the human c-fos promoter was characterized in vitro using HeLa nuclear and whole cell extracts. The c-fos gene and c-fos promoter fusions to the SV40 early region and the G-free cassette were active as templates, yielding faithfully initiated transcripts that were sensitive to alpha-amanitin. c-fos-SV40 and -G-free cassette templates were less efficiently transcribed when linearized, suggesting that template topology affected fos activity in vitro. Transcription conditions were optimized for both extracts using a mixture of the fos wild type promoter, a deleted fos promoter retaining just the TATA box, and the adenovirus major late promoter, all driving differently sized G-free cassettes. The fos promoter was inactive at low protein and DNA concentrations, and then showed a sharp rise in efficiency with a subsequent 2-fold increase in the amount of either DNA or protein. In contrast, high protein or DNA concentrations were required for the deleted fos promoter to show only weak activity. Preincubation of templates with extracts shortened the lag time before transcription could be detected, but did not lead to increased activity. By comparing reactions in the presence and absence of Sarkosyl, we estimate that only a subset of fos-driven templates was active, and that these templates were used for several rounds of transcription.