We have previously shown that in vitro culture of rat natural killer (NK) cells in high concentrations of recombinant interleukin 2 (rIL-2) leads to the expression of a surface glycoprotein with a molecular mass of approximately 42 kD. This glycoprotein, gp42, is not induced on other lymphocytes and thus provides a lineage-specific marker for rIL-2-activated NK cells. We here present the nucleotide sequence for gp42 cDNA. The open reading frame encodes 233 amino acids with three potential sites for N-linked glycosylation. The deduced amino acid sequence lacks an apparent transmembrane domain and instead contains a hydrophobic COOH terminus that is characteristic of glycosylphosphatidylinositol (GPI)-anchored surface proteins. Consistent with this, gp42 is cleaved from the NK-like cell line, RNK-16, by phosphatidylinositol-specific phospholipase C (PI-PLC), as is gp42 expressed on CHO cells that have been transformed with gp42 cDNA. On rIL-2-activated NK cells, gp42 is resistant to PI-PLC, though our studies suggest that gp42 on these cells is still expressed as a GPI-anchored molecule. Antibody to gp42 stimulates in RNK-16 cells an increase in inositol phosphates and in intracellular calciu, signals that are associated with the activation of lymphocytes, including NK cells. rIL-2-activated NK cells, however, lack this response to gp42 as well as to other stimuli. Thus, gp42, the only NK-specific activation antigen, is a GPI-anchored surface molecule with the capacity to stimulate transmembrane signaling.