We previously showed that an oncogenic Cbl mutant (70Z) is constitutively active in transcriptional activation of nuclear factor at activated T cells (NFAT). However, the mechanism underlying this effect remains unclear. Here we analyzed the effects of 70Z mutations at an amino-terminal loss of function site (Gly-306) and at carboxyl-terminal potential tyrosine or serine phosphorylation sites on association with signaling proteins and on NFAT activation. Mutation at Gly-306 of 70Z disrupted its association with Zap-70 and almost completely abolished its ability to induce NFAT activation under basal and ionomycin-stimulated conditions. However, mutations at potential tyrosine or serine phosphorylation sites had little effect. In fact, expression of 70Z with Tyr-700, Tyr-731, or Tyr-774 mutated to Phe increased NFAT activity in comparison with unmutated 70Z. These findings suggest that an amino terminus-mediated interaction of 70Z with Zap-70 plays a positive role and that a carboxyl terminus-mediated, phosphotyrosine-dependent interaction with their binding proteins plays a negative role in 70Z-mediated NFAT activation. In support of this notion are the observations that 70Z reduced T cell receptor-induced NFAT activation and that wild-type Cbl further inhibited this event, suggesting that both 70Z and wild-type Cbl employ a similar mechanism by which Cbl proteins dually regulate T cell receptor-mediated signaling.