Coproporphyrinogen oxidase (CPO) catalyzes the sixth step of the heme biosynthetic pathway. To assess the tissue-specific regulation of the CPO gene promoter, mouse genomic DNA clones for CPO were isolated. Structural analysis demonstrated that the mouse CPO gene spans approximately 11 kb and consists of seven exons, just like its human counterpart. Functional analysis of the promoter by transient transfection assays indicated that synergistic action between an SP-1-like element at -21/-12, a GATA site at -59/-54, and a novel regulatory element, CPRE (-GGACTA-CAG-) at -49/-41, is essential for the promoter activity in murine erythroleukemia (MEL) cells. In nonerythroid NIH3T3 cells, however, the GATA site is not required. Gel mobility shift assays demonstrated that specific DNA-protein complexes can be formed with each element, and that there are cell-specific differences in factors, which bind to the SP-1-like element between MEL and NIH3T3 cells. These results provide evidence for differential regulation of the promoter function of CPO gene between erythroid and nonerythroid cells.