Previously, we cloned a human cDNA encoding a protein which has a 92% amino acid sequence identity to a rat initiation factor-2 associated protein (p67). Rat p67 plays an important role in translational regulation by preventing the phosphorylation of the alpha subunit of initiation factor-2. Interestingly, several lines of indirect evidence suggested that this protein may also function as a methionine aminopeptidase (MetAP). To test this hypothesis, we expressed the human cDNA in a baculovirus system, purified it to homogeneity and characterized it. Using 13 different peptide substrates, we found that the human p67 has a similar substrate specificity with other MetAPs. Kinetic analyses revealed that the Kcat/K(m) values of the human MetAP on two representative substrates are similar to those of yeast and porcine MetAPs. Furthermore, we found that this enzyme, like other MetAPs, is also a cobalt-dependent metalloenzyme.