Elevation of intracellular cAMP levels in the human prostatic adenocarcinoma cell line LNCaP results in the induction of reversible neuroendocrine differentiation and cell growth arrest. We have used the differential display technique to identify genes that are differentially expressed during cAMP induced neuroendocrine differentiation in LNCaP cells. We identified the human ribosomal S3a gene as being down regulated in response to LNCaP differentiation. The S3a gene is known to be expressed at high levels in both tumors and cancer cell lines. It has also been shown that down regulation of S3a is associated with a loss of the transformed phenotype. In order to better ascertain the mechanism by which S3a gene expression is decreased during differentiation, the promoter region for this gene was analyzed. Electrophoretic mobility shift assay, antibody supershift assays, site-directed mutagenesis, and luciferase reporter gene analysis were employed to authenticate the roles of several transcription factors in the regulation of the S3a gene. We found that two cyclic AMP response elements, a Sp1 element, and a GA-binding protein element were involved in the regulation of S3a gene expression. The CRE elements were found to be necessary for high level expression of the 53a gene in undifferentiated LNCaP cells. Mutations in the CRE elements abolished CREB-1 binding and resulted in a 57% decrease in S3a gene expression. The addition of cAMP elevating agents to LNCaP cells in sufficient quantity to induce differentiation generated a 50% decrease in S3a gene expression. These results suggest that the CRE elements participate in cAMP-induced down regulation of gene expression. Furthermore, our experiments demonstrate that occupation of the GABP binding site results in a substantial decrease in S3a promoter activity.