Pituitary-derived trophic hormones regulate cell-type-specific expression of VL30 retrotransposons in tissues that are engaged in steroidogenesis. We show that adrenocorticotropic hormone and forskolin induced VL30 transcription in the steroidogenic adrenal cell line Y1 and that the transcriptional activation was cell type- and protein kinase A-dependent. Three novel cAMP-responsive elements (CREs), within the VL30 long terminal repeat, were identified and shown to activate transcription synergistically when templates bearing multiple sites were compared with templates bearing a single site. This type of regulation was evident only in forskolin-treated cells, and the response elements were found to be inactive as mediators of constitutive transcription. In vitro binding analyses indicated that a consensus CRE and the nonconsensus VL30 CREs differ with respect to binding affinity and specificity to a number of nuclear factors that were identified to be related to proteins within the CREB, Jun, and C/EBP families of transcription factors. The relatively low affinity and/or a restricted binding specificity of the VL30 CREs made it possible to detect forskolin-induced binding of CREB- and Jun-related proteins to these sequences. We suggest that cAMP-induced transcription, specific for steroidogenic cells, can be mediated by a novel type of nonconsensus CREs and that the mechanism for this type of gene regulation is distinct from that mediated through a consensus CRE. We also report the identification of a novel factor, distinct from previously characterized CRE-binding proteins, that constitutively binds to the identified CREs.