We previously showed that a peptide (PR1) derived from the primary granule enzyme proteinase 3 induced peptide specific cytotoxic T lymphocytes (CTL) in a normal HLA-A2.1+ individual. These CTL showed HLA-restricted cytotoxicity to myeloid leukemias (which overexpress proteinase 3). To further investigate their antileukemic potential, we studied the ability of PR1-specific CTL, derived from two HLA-A2.1+ normal individuals, to inhibit colony-forming unit granulocyte-macrophage (CFU-GM) from normal and leukemic individuals. CTL from 20 day PR1 peptide-pulsed lymphocyte cultures showed 89% to 98% HLA-A2.1-restricted colony inhibition of chronic myeloid leukemia targets. Colony formation in normal HLA-A2.1+ bone marrow or HLA-A2.1- CML cells was not inhibited. Sequencing of the exon encoding PR1 showed that colony inhibition was not caused by polymorphic differences in proteinase 3 between effectors and targets. Analysis by flow cytometry showed that proteinase 3 was overexpressed in the leukemia targets compared with normal marrow targets (median channel fluorescence 1,399 v 298, P = .009). These results show that PR1-specific allogeneic T cells preferentially inhibit leukemic CFU-GM based on overexpression of proteinase 3, and that proteinase 3-specific CTL could be used for leukemia-specific adoptive immunotherapy.