We describe the cloning, sequence analysis, and chromosomal mapping of a novel mitochondrial ATP synthase subunit 9cDNA, P3. Subunit 9 transports protons across the inner mitochondrial membrane to the F1-ATPase protruding on the matrix side, resulting in the generation of ATP. Sequence analysis of the P3 cDNA reveals only 80% identity with the human subunit 9 genes P1 and P2 in the DNA sequence encoding the mature peptide. However, this sequence predicts a mature protein identical to P1 and P2. The predicted sequence of the P3 leader peptide differs from the P1 and P2 leaders, but retains the "RFS" motif critical for mitochondrial import and maturation. The P3 gene (ATP5G3) maps to chromosome 2.