In order to characterize the CD3 zeta-related protein found in human natural killer (NK) cells and compare it with CD3 zeta expressed in T lymphocytes, the present study was performed. A polyclonal CD3-CD16+NK population displaying a strong non-major histocompatibility complex-restricted cytotoxic activity against the NK target K-562 was isolated and a product corresponding to CD3 zeta amplified using the polymerase chain reaction method. This 0.6-kb product was present in similar amounts in NK cells and T cells. In contrast, a product corresponding to CD3 delta was amplified from T lymphocytes exclusively. Thus, the CD3 zeta product detected in NK cells did not originate from contaminating T cells. DNA sequence analysis of two independent polymerase chain reaction products from the NK cells demonstrates that human NK cells and mature T cells share a CD3 zeta subunit with an identical primary amino acid sequence. The nucleotide sequence of a third NK-derived cDNA revealed an insertion of a CAG triplet encoding an additional glutamine residue in the cytoplasmic domain. Since this residue is encoded by nucleotides at a putative RNA splice junction, it possibly results from a difference in pre-mRNA splicing. Taken together, these data show that CD3 zeta is not structurally distinct in NK cells and in T lymphocytes.