Ku protein, a heterodimer of 70 kDa (Ku70) and 86 kDa (Ku86) polypeptides, is involved in non-homologous DNA end-joining (NHEJ) of DNA double-strand break repair and V(D)J recombination in combination with the catalytic component of DNA-dependent protein kinase (p470). Although Ku protein is known to be ubiquitously present in eukaryotic cells, it was previously reported to be absent in mature neutrophils. Using a mixture of protease inhibitors in the isolation procedure of neutrophils from human peripheral blood, we were able to detect Ku in the neutrophils by immunoblot and flow-cytometric analyses. Transcripts of Ku70 and Ku86 genes were also detected by the reverse transcriptase-polymerase chain reaction (RT-PCR), and Ku protein was shown to be localized in the nucleus of neutrophils as a heterodimer. Like poly(ADP-ribose) polymerase-1, neither mRNA nor protein of p470 was detected in the neutrophils. These results suggest that Ku is involved independently of p470 in DNA metabolism and signal transduction.