Plasma membrane V-ATPase isolated from midgut and Malpighian tubules of the tobacco hornworm, Manduca sexta, contains a novel prominent 20-kDa polypeptide. Based on N-terminal protein sequencing, we cloned a corresponding cDNA. The deduced hydrophobic protein consisted of 88 amino acids with a molecular mass of only 9.7 kDa. Immunoblots of the recombinant 9.7-kDa polypeptide, using a monoclonal anti- body to the 20-kDa polypeptide, confirmed that the correct cDNA had been cloned. The 20-kDa polypeptide is glycosylated, as deduced from lectin staining. Treatment with N-glycosidase A resulted in the appearance of two additional protein bands of 16 and 10 kDa which both were immunoreactive to the 20-kDa polypeptide-specific monoclonal antibody. Thus, extensive N-glycosylation of the novel Vo subunit M9.7 accounts for half of its molecular mass observed in SDS-polyacrylamide gel electrophoresis. M9.7 exhibits some similarities to the yeast protein Vma21p which resides in the endoplasmic reticulum and is required for the assembly of the Vo complex. However, as deduced from immunoblots as well as from activities of the V-ATPase and endoplasmic reticulum marker enzymes in different membrane preparations, M9.7 is, in contrast to the yeast polypeptide, a constitutive subunit of the mature plasma membrane V-ATPase of M. sexta.