Using a new mAb, 2F1, we characterize a mouse natural killer (NK) cell antigen termed 'killer cell lectin-like receptor G1' (KLRG1; formerly mouse MAFA or 2F1-Ag). KLRG1 is expressed on 30-60% of murine NK cells, and a small fraction of T cells, and is composed of a homodimer of glycosylated 30-38-kDa subunits. Strikingly, cell surface expression of KLRG1 by NK cells was substantially down-regulated in mice deficient for expression of class I molecules, in contrast to the Ly49 lectin-like NK receptors, which are up-regulated in class I-deficient mice. We could not demonstrate binding of KLRG1 to class I molecules in a cell-cell adhesion assay. Transgenic expression of KLRG1 under heterologous transcription elements was unaffected by class I deficiency, indicating that class I molecules do not affect the KLRG1 protein directly, and suggesting that regulation is at the level of expression of the endogenous KLRG1 gene. Evidence is presented that class I molecules regulate KLRG1 via interactions with class I-specific inhibitory Ly49 molecules and SHP-1 signaling. Thus, although KLRG1 and Ly49 molecules are both lectin-like inhibitory receptors that are regulated by class I expression, the effects of class I on the cell surface expression of the molecules are opposing, and the underlying regulatory mechanisms are distinct.