A 75-kDa melanosomal glycoprotein (gp75) is the product of a gene that maps to the b (brown) locus, a genetic locus that determines coat color in the mouse. The b locus is conserved (88% identity) between mouse and human. The mouse monoclonal antibody TA99 was used to study the biosynthesis and processing of gp75. gp75 was synthesized as a 55-kDa polypeptide, glycosylated by addition and processing of five or more Asnlinked carbohydrate chains through the cis and trans Golgi, and transported to melanosomes as a mature 75kDa form. Synthesis and processing of gp75 was rapid (T 1 2< 30 min), and early steps in processing were required for efficient export of gp75 to melanosomes. Fully processed mature gp75 was quite stable (T 1 2= 22–24 h) in the melanosome. Digestion of high-mannose carbohydrate chains with endo-β-N-acetylglucosaminidase H revealed two alternative processed forms of gp75 that differed in the number or composition of complex-type carbohydrate chains. The rate of synthesis and movement through intracellular membrane compartments was the same for both glycosylated forms. Studies with inhibitors of steps in oligosaccharide processing showed that alternative forms of gp75 were generated during trimming reactions by mannosidase IA/IB and that further maturation resulted in the two mature forms of gp75. We propose that the kinetics of biosynthesis and processing reflect events in the biogenesis and maturation of melanosomes.