Using the CD4⁺ human T cell clone P28, we demonstrated that the HIV‐1 glycoprotein gp120 inhibited CD3‐induced inositol trisphosphate production, calcium influx and T cell proliferation. Additionally, gp120 was shown to dissociate the tyrosine kinase p56^lck from CD4 in CEM cells, with a concommittant inhibition of CD4‐linked kinase activity. We have addressed the question whether disruption of CD4/p56^lck or CD4/CD3‐T cell receptor interactions, or both, could account for the inhibitory effect of gp120 in P28 cells. By comparing the effects of various anti‐CD4 monoclonal antibodies (mAb) with those of gp120, we show that gp120 and IOT4a modulate CD4 expression, and decrease CD4‐associated p56^lck and CD4‐linked kinase activity at the plasma membrane. In contrast, OKT4A and OKT4 anti‐CD4 mAb have no inhibitory effect. Interestingly, gp120 also inhibits CD3‐induced Lck activation and cellular tyrosine phosphorylation, particularly of phosphoinositide‐specific phospholipase C‐γ‐1. Kinetic experiments reveal that the inhibitory effect of gp120 on CD3‐induced tyrosine phosphorylation appears as early as 30 min, but culminate when CD4‐p56^lck complexes disappear from the cell surface after 4 h. These results suggest that a negative signal is triggered by gp120 that results, after a few hours, in down‐modulation of CD4‐p56^lck complexes and the impairment of CD3 signaling. Supporting this hypothesis, gp120 inhibits CD3‐linked kinase activity as shown by the inhibition of the phosphorylation of CD3 chains, leading to the inhibition of subsequent signal transduction.