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. J. Leukoc. Biol. 87: 815-822; 2010.