We have previously reported that ATPgammaS, a slowly hydrolyzed analog of ATP, inhibits the activation of human CD4(+) T lymphocytes by anti-CD3 and anti-CD28 mAb. In this report we have partially characterized the signaling mechanisms involved in this immunosuppressive effect. ATPgammaS had no inhibitory effect on CD4(+) T-cell activation induced by PMA and anti-CD28, indicating that it acts proximally to the TCR. It had no effect on the calcium rise induced by CD3/CD28 stimulation, but inhibited the phosphorylation of three kinases, ERK2, p38 MAPK and PKB, that play a key role in the activation of T cells. The receptor involved in these actions remains unidentified.