Aminopeptidase P (APP) isoforms specifically remove the N-terminal amino acid from peptides that have a proline residue in the second position. The mRNA levels of three different isoforms, each coded by a different gene, were determined in 16 human tissues and in peripheral blood mononuclear cell (PBMC) fractions by RT-PCR. The cytosolic isoform, APP1, and the cell surface membrane-bound isoform, APP2, are expressed in all of the human tissues and PBMC fractions examined. The very high expression of APP2 mRNA in kidney compared to other tissues was confirmed by enzyme activity measurements. Among the PBMC fractions, APP2 expression is highest in resting CD8(+) T cells, but decreases in these cells following their activation with phytohemagglutinin; in contrast, expression of APP2 increases in CD4(+) T cells upon activation. The third isoform, APP3, is a hypothetical protein identified by nucleotide sequencing. A detailed analysis of its amino acid sequence confirmed that the protein is an aminopeptidase P-like enzyme with greater similarity to Escherichia coli APP than to either APP1 or APP2. Two splice variants of APP3 exist, one of which is predicted to have a mitochondrial localization (APP3m) while the other is cytosolic (APP3c). Both forms are variably expressed in all of the human tissues and PBMC fractions examined.