CD69 is the earliest activation antigen expressed on T lymphocytes upon stimulation through the TCR, or with stimuli that mimic TCR triggering. Here we describe that the phorbol ester PMA and a calcium ionophore had a synergistic effect on both CD69 antigen expression and promoter activity in Jurkat cells, that was sensitive to cyclosporin A (CsA). CD69 promoter analysis indicated that the sequence -78 to +16 contained the elements responsible for PMA and PMA plus calcium ionophore induction, as well as CsA inhibition. Mutagenesis of two previously described AP-1 motifs did not affect either the basal or the inducible promoter activities. Electrophoretic mobility shift assays allowed the identification of three novel inducible complexes composed by Egr-1/Egr-3, Egr-1, and ATF-3/Fos. Mutation of each sequence resulted in a partial reduction of the basal promoter activity, whereas the inducibility by PMA plus calcium ionophore remained almost unaffected. It was necessary to combine at least two mutations to obtain a significative or complete reduction of the response to the mitogenic stimulus. These results indicate that the inducible expression of CD69 gene by mitogenic signals is regulated by multiple cis-acting elements and by the interplay of transcription factors of the AP-1, EGR and ATF/CREB families.