Mitochondria have a receptor complex in the outer membrane which recognizes and translocates mitochondrial proteins synthesized in the cytosol. We report here the identification and functional analysis of human Tom22 (hTom22). hTom22 has an N-terminal negatively charged region exposed to the cytosol, a putative transmembrane region, and a C-terminal intermembrane space region with little negative charge. Tom22 forms a complex with Tom20, and its cytosolic domain functions as an import receptor as in fungi. An import inhibition assay, using pre-ornithine transcarbamylase (pOTC) derivatives and a series of hTom22 deletion mutants, showed that the C-terminal segment of the cytosolic domain is important for presequence binding, whereas the N-terminal domain is important for binding to the mature portion of pOTC. No evidence for pOTC interaction with the Tom22 intermembrane space domain was obtained. Binding studies revealed that the presequence is critical for pOTC binding to Tom20, whereas both the presequence and mature portion are important for binding to Tom22. A cell-free immunoprecipitation assay indicated that an internal segment of the Tom22 cytosolic domain is important for interaction with Tom20.