We have recently described the A6H antigen as a novel 120-140 kDa molecule which is co-expressed on human peripheral blood T cells and renal cell carcinoma cells. Engagement of the A6H antigen results in co-stimulation of CD4+ T cells but it remained unknown how cross-talk between the A6H antigen and the TCR-CD3 complex takes place and which signaling pathway might be involved. Here we show that ligation of the A6H antigen with mAb induces tyrosine phosphorylation of the Lck protein tyrosine kinase (PTK). Co-ligation of the A6H antigen with CD3 resulted in augmented Lck phosphorylation and mitogenesis. In addition, A6H ligation induced an up-regulation of CD3-mediated phosphorylation of the 23 kDa high mol. wt form of TCR zeta and the zeta-associated protein, ZAP-70. Co-precipitation of Lck and ZAP-70 was only seen in T cells activated by combined A6H and anti-CD3 stimulation. In contrast, another Src family PTK, Fyn, was not affected by A6H ligation. In conclusion, we now demonstrate, for the first time, that A6H ligation triggers Lck phosphorylation, and that cross-talk between A6H and the TCR-CD3 complex involves Lck, ZAP-70 and the slow migrating isoform of TCR zeta. These results further suggests that A6H ligation is sufficient for triggering some of the early events in T cell activation, whereas full activation of the T cell, characterized by proliferation and cytokine production, requires co-ligation of the TCR-CD3 complex.