The effect of phorbol esters on transcription of human type 5 adenovirus (Ad5) early region 1 (E1) genes was studied in two human cell lines (293 and KB16) that contain integrated viral DNA. In 293 cells 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol 12,13-dibutyrate and phorbol 12,13-didecanoate caused a 2- to 3-fold increase in cytoplasmic levels of E1 RNA within 90 min, whereas the nonpromoting TPA analogues 4-alpha-phorbol 12,13-didecanoate and phorbol, or the addition of serum to serum-starved cells, had no effect on E1 RNA. Stimulation by TPA was transient, and the concentrations of all E1 RNA species returned to basal levels by 5 h. The kinetics of E1 RNA accumulation in the presence of TPA were unaffected by cycloheximide, although levels of E1 RNA remained elevated in these conditions. The apparent molecular sizes of major RNA species synthesized from the E1A and E1B regions in 293 cells were the same as those seen early in productive infection by Ad5, and were not changed after induction by TPA. Nuclear run-on assays showed that E1 transcription, as well as transcription of c-fos, c-myc, and beta-actin was stimulated in 293 cells within 30 min of TPA exposure and returned to basal levels by 60 min. In contrast to the 293 cells, expression of Ad5 E1 genes in a second transformed human cell line, KB16, was not altered by TPA. These results show that the induction of Ad5 E1 genes by TPA does not require their presence on infecting genomes, but suggest that inducibility of integrated, functionally expressed E1 genes can be influenced by factors other than their primary sequence.