The mouse- and rat-platelet-specific hamster monoclonal antibody (MAb) 1C2, previously found to react with a thrombin-sensitive 74-kD glycoprotein, was now shown to recognize platelet glycoprotein V (GPV, CD42d). 1C2 reacted with NIH-3T3 cells in which recombinant mouse or rat GPV was expressed. Both 1C2 and 4A5, another mouse-platelet-specific rat MAb, immunoprecipitated GVP, although they recognized different epitopes. Side-by-side comparison confirmed that 1C2 as well as RPM.9, a MAb against rat GPV, recognized the same rat platelet molecule. In a mouse bone marrow culture, 1C2+ megakaryocytes emerged from CD41 (GPIIb)+1C2- megakaryocytes. Because 1C2+ megakaryocytes exhibited higher DNA ploidy distribution than CD41+ cells, GPV likely appears in the late stage of megakaryocyte maturation. This study established 1C2 as a MAb against mouse and rat GPV, namely CD42d, and as useful tool to study rodent megakaryopoiesis.