To understand the mechanism by which phorbol esters (PMA) stimulate c-jun transcription in human leukemic cell line U937, we have mutated specific enhancer sequences within the c-jun promoter. We find in the region of DNA from -132 to +170 containing Sp1, C-TF and AP-1 sequences that mutation of the AP-1 sequence alone is not sufficient to abrogate transcription, and mutation of the Sp1 sequence increases transcription 4-fold. Although mutation of the CTF site had no effect, CTF and AP-1 mutations together totally abrogate PMA-induced transcription. In comparison mutations of either of these sites alone or together in a construct containing -1639/+740 of the c-jun promoter had no effect on transcription. Because this data suggested the possibility of other upstream control regions, we sequenced the promoter from -142 to -1639. This sequence demonstrates a greater than 70% homology between human, and mouse c-jun promoters for the region from -142 to -441, and a second AP-1-like site in the -183 to -192 region. Mutation of this site did not influence transcription by PMA. By making constructs containing varying portions of the promoter, we have identified the region between -142 and -711 to be responsible for mediating PMA-induced c-jun transcription.