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The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross\u2010reaction with rat peritoneal macrophages (M\u03c6). We identified the antigen molecule for 9F5: the 50\u2010 to 70\u2010kDa fragments of rat glycoprotein nonmetastatic melanoma protein B (GPNMB)\/osteoactivin, which started at Lys<jats:sup>170<\/jats:sup>. In addition, 9F5 immunoreactivity with GPNMB depended on the activity of furin\u2010like protease(s). More important, rat type 1 MG expressed the GPNMB fragments, but type 2 MG and M\u03c6 did not, although all these cells expressed mRNA and the full\u2010length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and M\u03c6, may have furin\u2010like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double\u2010immun\u03bfstaining with 9F5 antibody and anti\u2010Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist <jats:italic>in vivo<\/jats:italic>. We propose that 9F5 is a useful tool to discriminate between rat type 1 MG and other subtypes of MG\/M\u03c6 and to reveal the role of the GPNMB fragments during developing brain. 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