The human embryonal carcinoma cell line NEC14 can be induced to differentiate by the addition of N,N'-hexamethylene-bis-acetamide (HMBA). After treatment with HMBA, the level of cyclin A transcript decreased steeply, reaching less than one-tenth of the original level by 48 h. The promoter elements concerned with this down-regulation were studied by using reporter genes and by analyzing DNA-protein complexes. The deletion of the sequence between -608 and -259 containing three GC boxes decreased the promoter activity to about a half, and further deletion up to -194, eliminating the ATF/CRE site, resulted in a decrease to about a tenth of the original level in undifferentiated NEC14 cells. These sequences were involved in down-regulation of the promoter activity in differentiation-induced NEC14 cells. DNA-protein complexes formed at the ATF/CRE site with extracts prepared from undifferentiated and differentiation-induced cells gave the same footprint, but showed different electrophoretic mobilities. The supershift assay with specific antibodies against ATF-1 and ATF-2 indicated that both factors were depleted in the complex after induction of NEC14 cell differentiation. Both the ATF/CRE site and GC boxes seemed to be also involved in up-regulation of the cyclin A promoter in growth-stimulated human fibroblasts at the G1/S boundary.