Crosslinking of the CD4 coreceptor can inhibit subsequent T-cell activation via the T-cell antigen receptor (TCR)/CD3 complex. The ability of the human immunodeficiency virus (HIV) envelope protein, gp 120, to cause similar inhibition has implicated this inhibitory signal in the induction of T-cell anergy and apoptosis observed in the acquired immunodeficiency syndrome (AIDS). In order to clarify the biochemical basis of this inhibition, we analyzed the effect of CD4 ligation on early signaling events induced by subsequent CD3×CD4 co-crosslinking. By comparison with CD3 crosslinking alone, CD3×CD4 co-crosslinking of a CD3+CD4+ human T-cell leukemia line (SupT1) resulted in an enhanced increase in free intracellular calcium concentration and tyrosine phosphorylation of several cellular substrates, including the prominent phosphorylation of an unidentified 120-kDa protein (p120). Prior CD4 ligation inhibited these responses. Similar results were obtained with A3.01, another CD3+CD4+ T leukemic line. However, p120 was only minor phosporylated on tyrosine upon receptor crosslinking in A2.01/CD4 (-cyt401), a derivative line expressing a truncated CD4 coreceptor lacking its cytoplasmic domain which binds the p56^lck protein tyrosine kinase (PTK). Furthermore, prior CD4 ligation failed to inhibit in this line the increased tyrosine phosphorylation induced by subsequent CD3 × CD4 co-crosslinking. Thus, prior CD4 crosslinking, or expression of truncated CD4, are both associated with reduced p 120 phosphorylation. These results suggest that p 120 is a p56^lck substrate playing an important role during T-cell activation.