To study the transcriptional regulation of the vasopressin gene in vitro, 3 kb of the 5' regulatory region of the rat vasopressin gene was isolated and subcloned, along with a series of various deletion mutants, into vectors containing the luciferase reporter gene. After transfecting these genes transiently into the human choriocarcinoma cell line JEG-3 along with a glucocorticoid receptor (GR) expression vector, transcriptional activity was quantitated using the luciferase assay. Forskolin, 8-bromo-cAMP, and protein kinase A catalytic subunit expression all markedly increased transcription from the 3-kb promoter. Analyses with deletion mutants of the promoter showed that two cAMP-responsive element (CRE)-like sequences (-227 to -220 bp and -123 to -116 bp) contribute to this positive regulation. Expression of KCREB, a dominant negative mutant of the cAMP-responsive element binding protein (CREB), suggested the involvement of CREB. Transfection of the activator protein 2 (AP2) DNA consensus sequence partially blocked transcription. Dexamethasone suppressed forskolin-stimulated expression. The negative effect of glucocorticoid was GR dependent and may be mediated by a mechanism not involving GR binding to DNA because it was independent of the putative glucocorticoid-responsive element previously reported in the vasopressin promoter (-622 to -608 bp) and was preserved in the shorter promoter constructs in which no glucocorticoid-responsive element-like sequence was found. Our data suggest that several trans-acting factors including CREB, AP2, and GR are likely to be involved in vasopressin gene transcription and that the positive and negative regulation of vasopressin gene transcription is complex.