The human CGL-1/cytotoxic serine protease B gene (CSP-B; also known as granzyme B) is transcriptionally activated during cytotoxic T- lymphocyte maturation. Activation can be mimicked in the PEER T-cell leukemia cell line by treatment with 12-O-tetradecanoylphorbol-13- acetate (TPA) and N6–2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate (bt2cAMP). In this report, we show that a consensus AP-1 element and a consensus cAMP response element (CRE) located 5′ to the CSP-B transcriptional start site are both required for transcriptional activation of the CPS-B promoter in TPA + bt2cAMP-stimulated PEER cells. A 94-bp fragment containing both elements activates a heterologous promoter in an orientation-independent fashion. Several single nucleotide substitutions in the AP-1 site abolish activity of the 94-bp fragment. Several point mutations in the consensus CRE substantially reduce promoter activity, but one CRE mutation increases activity fourfold. Replacement of the CRE with a second copy of the AP- 1 site results in a level of transcriptional activity comparable with that of the wild-type sequence, but replacement of the AP-1 site with a CRE abolishes activity. Neither the AP-1 site nor the CRE can be effectively replaced with an SP-1 site. Deletions between the AP-1 site and the CRE retain full activity only if helical spacing is preserved, suggesting that synergism between these two elements is either the result of cooperative binding of factors to the DNA or of cooperative binding of DNA-bound factors to another protein.