Binding of CTL to MHC class I or fibronectin is activated through TCR signaling. Once activated, CTL adhesion to MHC class I results in tyrosine phosphorylation of CTL substrates, phosphatidylinositol (PI) turnover, and degranulation. Although activated adhesion to fibronectin does not itself initiate PI hydrolysis or degranulation, these responses are amplified once they become activated. In the present study we have examined the effect of cis unsaturated fatty acids (FA) and phenylarsine oxide (PAO) on CD8-mediated adhesion of CTL to immobilized class I protein and on biochemical and functional events that are triggered by this adhesion. Previous studies have shown that FA and low concentrations of PAO inhibit specific tyrosine phosphorylation events and degranulation but have no effect on PI turnover or CTL-target cell conjugates. The present results show that pretreating CTL with cis unsaturated, but not saturated, FA and low concentrations of PAO (< 0.5 microM) inhibit soluble anti-TCR-triggered binding of CD8 to immobilized MHC class I, tyrosine phosphorylation of CTL substrates, PI turnover, and degranulation. Addition of cis unsaturated FA or PAO after CTL have been allowed to bind to immobilized class I protein did not affect the level of adhesion. In contrast, neither cis unsaturated FA nor PAO affected the TCR-activated binding of CTL to fibronectin. These results suggest that activation of adhesion to the class I and fibronectin ligands involves divergent different pathways that can be distinguished by the FA and PAO agents.