We have identified a cDNA encoding a novel isoform of the mouse V-ATPase d subunit (d2). The protein encoded is 350 amino acids in length and shows 42 and 67% identity to the yeast d subunit (Vma6p) and the mouse d1 isoform, respectively. Reverse transcriptase-PCR analysis using isoform-specific primers demonstrate that d2 is expressed mainly in kidney and at lower levels in heart, spleen, skeletal muscle, and testis. Although d1 and d2 show similar levels of sequence homology to Vma6p, only the d1 isoform can complement the phenotype of a yeast strain in which VMA6 has been disrupted when cells are grown at 30 degrees C. The d2 isoform, however, can complement the vma6Delta phenotype when cells are grown at 25 degrees C. Moreover, partial assembly of the V-ATPase complex on the vacuolar membrane can be detected under these conditions, although assembly is significantly lower than that observed for the strain expressing Vma6p. This reduced assembly is also reflected in a reduced level of concanamycin-sensitive ATPase activity and proton transport in isolated vacuoles. Comparison of the kinetic properties of V-ATPase complexes containing Vma6p and d1 demonstrate that although the Km for ATP hydrolysis is similar (0.26 and 0.31 mm, respectively), the coupling ratio (proton transport/ATP hydrolysis) is approximately 3-6-fold higher for d1-containing complexes than for Vma6p-containing complexes. These results suggest that subunit d may play a role in coupling of proton transport and ATP hydrolysis.