CRF, in addition to its role in the hypothalamus, demonstrates species-specific expression in the placentas of higher primates, but not rodents. Transient transfections of BeWo and JEG-3 choriocarcinoma cells, as models for human trophoblasts, demonstrate regulated expression of human (h) CRF-luciferase reporter genes, whereas little or no expression is detected in other lines, including CV-1 cells. The rodent choriocarcinoma cell line, Rcho-1, a model for rodent trophoblasts, is defective in the expression of transfected hCRF genes. The mouse CRF promoter behaves similarly to the corresponding hCRF construct. It is active in BeWo and inactive in Rcho-1 cells. The transcriptional response to cAMP contributes to the specific expression of CRF. Analyses of deleted or mutated hCRF promoters identify a key role for protein kinase A-dependent pathways. A major part, but not all, of this effect is mediated by the canonical cAMP response element conserved in mouse, rat, and human CRF promoters. Additional deletions of the human CRF promoter identify control regions that also contribute to the observed species-specific expression pattern, and each identified region binds factors in nuclear extracts derived from the appropriate cell line. These studies using human and rodent choriocarcinoma cell lines as models of placental trophoblasts demonstrate dominant effects of cellular trans-acting factors, rather than DNA sequence differences, in dictating the species-specific placental expression of CRF.