DGKγ (diacylglycerol kinase γ) was reported to interact with β2-chimaerin, a GAP (GTPase-activating protein) for Rac, in response to epidermal growth factor. Here we found that PMA and H2O2 also induced the interaction of DGKγ with β2-chimaerin. It is noteworthy that simultaneous addition of PMA and H2O2 synergistically enhanced the interaction. In this case, PMA was replaceable by DAG (diacylglycerol). The β2-chimaerin translocation from the cytoplasm to the plasma membrane caused by PMA plus H2O2 was further enhanced by the expression of DGKγ. Moreover, DGKγ apparently enhanced the β2-chimaerin GAP activity upon cell stimulation with PMA. PMA was found to be mainly required for a conversion of β2-chimaerin into an active form. On the other hand, H2O2 was suggested to induce a release of Zn2+ from the C1 domain of β2-chimaerin. By stepwise deletion analysis, we demonstrated that the SH2 (Src homology 2) and C1 domains of β2-chimaerin interacted with the N-terminal half of catalytic region of DGKγ. Unexpectedly, the SH2 domain of β2-chimaerin contributes to the interaction independently of phosphotyrosine. Taken together, these results suggest that the functional link between DGKγ and β2-chimaerin has a broad significance in response to a wide range of cell stimuli. Our work offers a novel mechanism of protein–protein interaction, that is, the phosphotyrosine-independent interaction of the SH2 domain acting in co-operation with the C1 domain.