Natural killer (NK) cell functions are regulated by a delicate balance of signals received through activating and inhibitory receptors expressed on the cell surface. Lectin-like transcript-1 (LLT1), expressed on a subpopulation of NK cells and other immune cells is a ligand for the NK cell inhibitory receptor, NKR-P1A (CD161). Previous studies showed that cross-linking surface LLT1 with a monoclonal antibody stimulated NK cell IFN-γ secretion but had no effect on cytotoxicity. Here, we have examined the signalling pathways associated with LLT1-stimulated IFN-γ secretion. We ligated LLT1 on NK92 cells with CD161 on target cells and analysed IFN-γ production in the presence of pharmacological inhibitors specific for various signalling mechanisms. These results indicate that LLT1 employs Src-PTK, p38 and ERK signalling pathways, but not PKC, PI3K or calcineurin. Phosphorylation studies of the signalling adaptor molecules confirmed that the ERK signalling pathway is associated with LLT1-mediated IFN-γ production. LLT1 ligation is not associated with any change in detectable IFN-γ mRNA levels suggesting that LLT1-stimulated IFN-γ production in NK cells may involve post-transcriptional or translational events.