The effect of prothymosin a on transcriptional elongation has been examined. The addition of prothymosin a to COS-l and NIH3T3 cell nuclei engaged in run-on transcription stimulated RNA synthesis approximately two- to threefold in a dose-dependent manner. Polyglutamic acid or a random polypeptide composed of glutamic acid, alanine, and tyrosine, did not substitute for prothymosin a. Enhanced transcription occurred in the presence of high and low doses of actinomycin D and in the presence of cu-amanitin, but not in nuclear extracts. The stimulatory effect was dependent on a limiting concentration of one nucleoside triphosphate and was nearly abrogated by saturating levels of precursors. In the presence of Sarkosyl, which itself increases transcription, prothymosin a was almost ineffectual. The data are consistent with a model in which prothymosin a does not interact directly with polymerases but, instead, nonspecifically decreases the barriers to diffusion of charged molecules in electrostatically charged environments.