Abstract Src family kinases are normally inhibited by phosphorylation of their C-terminal tail by the tyrosine kinase Csk, and are positively regulated by the phosphatase CD45 and by receptor stimulation. Attempts to study the role of Csk in T cell receptor (TCR) signaling have been hampered by early embryonic lethality of Csk-/- mice and by perturbed thymocyte development in LckCre x Cskfl/fl mice. We have established a small-molecule inhibitor system to determine the effects of Csk localization and catalytic function on the regulation of TCR signaling. A novel analog-sensitive allele of Csk, CskAS, maintains normal function, but can be rapidly and specifically inhibited by 3IB-PP1, an analog of the PP1 kinase inhibitor. The function of wild-type Csk is unaffected by 3IB-PP1. We show that membrane-targeted CskAS, but not cytoplasmic CskAS, blocks TCR signal transduction. Using 3IB-PP1 to inhibit CskAS leads to rapid activation of proximal T cell signaling events including phosphorylation of CD3ζ and ERK1/2, increased cytoplasmic calcium, and downstream upregulation of CD69 expression and downregulation of the TCR. Together, these data suggest that introduction of CskAS alleles are sufficient to alter the basal steady state of Src family kinase activity in T cells. Inhibition of CskAS thus releases cells from tonic inhibition, resulting in rapid T cell activation, even in the absence of stimulation through the antigen receptor. This chemical-genetic approach of specific Csk inhibition is a useful means of studying the dynamic inhibition of TCR signaling and may be useful for manipulating the function of T cells. Research support: JRS, NIH T32 Training Grant; CZ, NF1 Foundation; KMS, HHMI; AW, HHMI and the Rosalind Russell Medical Research Center for Arthritis