Oxa1p is a mitochondrial protein reported to be involved in the assembly of the cytochrome oxidase complex. In the absence of a functional Oxa1p, subunit II of the cytochrome oxidase accumulates as its precursor form (pCoxII). Using mitochondria isolated from a yeast strain bearing a temperature sensitive mutation in the Oxa1p, pet ts1402, we have analyzed the function of the Oxa1p protein. We demonstrate that the accumulation of pCoxII in the pet ts1402 mitochondria does not reflect a compromised Imp1p activity in this mutant. Furthermore, measurement of the membrane potential has shown it to be sufficient to support the export of CoxII from the matrix. Rather, we found that newly synthesized pCoxII accumulates in the matrix of the pet ts1402 mitochondria, because export across the inner membrane is inhibited in the pet ts1402 mitochondria. In conclusion, Oxa1p mediates the export of the N- and C-termini of the mitochondrially encoded subunit II of cytochrome oxidase from the matrix to the intermembrane space.