The cellular kinase complex P-TEFb is composed of Cdk9 and cyclin T, and it is required for expression of most protein-coding genes by RNAP II. Cdk9 has been shown recently to be activated in cis by autophosphorylation of Thr186 in its T-loop. Using a phosphospecific Cdk9 antibody, we examined the level of Cdk9 T-loop phosphorylation in resting and activated CD4(+) T lymphocytes. Cdk9 T-loop phosphorylation was found to be low-to-undetectable in resting CD4(+) T lymphocytes, and upon activation by distinct stimuli, there is a rapid (<1 h) increase in pCdk9 that does not require protein synthesis. The low level of Cdk9 T-loop phosphorylation was not to be a result of the absence of an associated regulatory cyclin partner. These observations suggest that autophosphorylation of the Cdk9 T-loop is repressed in resting CD4(+) T lymphocytes. The low level of T-loop phosphorylation in resting cells is also reflected in a low level of phosphorylation of Ser2 in the carboxyl terminal domain of RNAP II, suggesting that lack of Cdk9 T-loop autophosphorylation may limit RNAP II elongation in quiescent CD4(+) T lymphocytes.