We have examined the 5′‐flanking region (944 bp) of the human choline acetyltransferase (hChAT) gene for sequences that modulate its transcriptional activity and identified a sequence 5′‐TGACCA‐3′ which confers c‐Jun/c‐Fos (AP‐1) inducibility of homologous and heterologous promoters. Using transient transfections in neuroblastoma NE‐1–115 and COS‐1 cells, we show that ligand‐activated estrogen receptor (HEGo) represses the transcriptional activation by c‐Fos/c‐Jun. Testing HEGo mutants in transfection assays reveals that the ligand‐binding domain is crucial for this repression, whereas the N‐terminal (A/B) region and the DNA‐binding domain are not essential. Gel retardation assays show that the hChAT AP‐1 recognition sequence binds in vitro baculovirus‐produced c‐Jun/c‐Fos proteins. This binding is inhibited by addition of baculovirus‐produced HEGo. In contrast to HEGo, ligand‐activated glucocorticoid, androgen, and retinoic acid receptors (RARs) enhance the transcription activation induced by c‐Jun/c‐Fos. All three types of RARs—RARα, β, γ—and RXRα are able to stimulate AP‐1 activity on the proximal hChAT promoter. Several mechanism possibilities involving protein‐protein interaction are discussed to explain the phenomena. © 1995 Wiley‐Liss, Inc.