The biosynthesis of the erythrocyte anion transport protein (Band III) was studied in erythroid precursor cells obtained from the spleens of anemic mice. Newly synthesized Band III was inserted during or immediately after translation into rough endoplasmic reticulum membranes. The asymmetric orientation of Band III in these membranes resembled that of mature Band III in erythrocyte membranes, with the NH2-terminal portion of the molecule facing the cytoplasm. At this stage Band III contained a high mannose core oligosaccharide, which was susceptible to cleavage by endoglycosidase H. During the next 20 to 30 min, this oligosaccharide was processed to a form resistant to endoglycosidase H degradation, presumably in the Golgi complex. The processed Band III was subsequently expressed on the cell surface, at about 30 to 45 min after synthesis. In many respects, therefore, the biosynthesis of Band III resembles that of cotranslationally inserted proteins whose NH2-terminal portions are exposed on the exterior of the cell, like VSV glycoprotein, HLA-A antigens, and glycophorin.