Previous studies indicate that a human chorionic somatomammotropin (hCS) gene enhancer (CSEn) associated with the growth hormone (hGH) gene locus is involved in directing cell-specific expression of the hCS genes in placenta. In the current studies, we report a detailed structural analysis of this enhancer. CSEn stimulated transcription of a variety of promoters, including the hCS, human growth hormone, thymidine kinase, and Rous sarcoma virus promoters, in human choriocarcinoma cell lines (BeWo and JEG-3) but not HeLa cells or rat somatolactotrophes (GC). Maximal enhancer activity was confined to a 242-base pair DNA segment. Of several CSEn subfragments, only the En 57/242 subfragment retained activity (33.5% wild-type). The CSEn DNA sequence contained direct and inverted repeat motifs and sequences related to the SV40 enhansons, GT-IIC, GT-I, and SphI/SphII. DNase I footprint analysis revealed that most of these sites were protected by nuclear proteins derived from BeWo, JEG-3, HeLa, and GC cells. Site-specific block mutation of the GT-IIC-related and inverted repeat motifs virtually abolished enhancer activity, and mutation of all but the GT-I-related motif resulted in significant loss (30-60%) of activity. These data demonstrate that the CS enhancer is comprised of multiple elements related to SV40 enhansons that interact cooperatively to generate enhancer function.