ATP11 is a nuclear gene product that is required for assembly of mitochondrial F1-ATPase in the yeast Saccharomyces cerevisiae. ATP11 is synthesized in the yeast cytoplasm with an N-terminal targeting sequence. Following import into mitochondria, the leader sequence is cleaved, generating the functional form of the protein. ATP11 is present in small amounts in yeast mitochondria, which has made it difficult to study its role in F1 assembly. We have developed a bacterial expression system for the overproduction of the mature form of ATP11 and its biotinated derivative, BTATP11. Yeast complementation assays showed that the DNA fragments used to produce ATP11 and BTATP11 in bacteria encode biologically active proteins. The recombinant proteins produced in bacteria were purified to homogeneity and their physical characteristics were shown to be similar to those of the mitochondrial ATP11 protein synthesized in yeast.