We have characterised a 16.3-kDa human protein that functions as a receptor for the import of preproteins into mitochondria. Based on amino acid sequence alignments, the protein (hMas20p) is 41% similar to Mas20p (20-kDa mitochondrial assembly protein) from yeast Saccharomyces cerevisiae and 38% similar to MOM19 (19-kDa mitochondrial outer-membrane protein) from Neurospora crassa. hMas20p has a putative N-terminal transmembrane sequence of 29 amino acids and an acidic C-terminus. A 13-kDa fragment [des-(1-29)-hMas20p], which lacks the 29-amino acid putative N-terminal transmembrane domain, is soluble when expressed in Escherichia coli. Antibodies produced against this domain crossreacted with a protein of 16 kDa in outer membranes of mitochondria from rat liver and inhibited import of protein into isolated mitochondria from rat liver. In addition, the recombinant soluble domain folds into a functional structure as it competes with hMas20p on the mitochondrial surface for precursor binding, confirming the functional role of hMas20p in the import of preproteins into mitochondria.