Csf1r mRNA in adult mice is expressed in cells of the macrophage lineage, and during development, it is also expressed from a separate promoter in placental trophoblast cells. This mouse trophoblast promoter sequence is conserved across species, but human trophoblasts actually initiate transcription from a separate promoter 20 kb upstream, which is not conserved in rodents. A 7.2-kb fragment of the mouse Csf1r genomic DNA, including the 3.5-kb promoter, the first coding exon and downstream intron, is sufficient to direct reproducible position- and copy number-independent expression of an EGFP reporter in vitro and in vivo. In this study, we have examined the consequence of removal of the 150-bp fragment encompassing the conserved trophoblast promoter region in the context of the 7.2-kb promoter on reporter gene expression in transgenic mice. The deletion ablated expression in the placenta but also abolished expression in multinucleated OCL and reduced expression in macrophages. RT-PCR analyses of Csf1r mRNA revealed that mouse OCL use another promoter within this region, distinct from that used in placental trophoblasts, to generate an alternative 5'UTR.