Protein kinase C (PKC) (cid:2) is an established component of the immunological synapse and has been implicated in the control of AP-1 and NF- (cid:4) B. To study the physiological function of PKC (cid:2) , we used gene targeting to generate a PKC (cid:2) null allele in mice. Consistently, interleukin 2 production and T cell proliferative responses were strongly reduced in PKC (cid:2) -deficient T cells. Surprisingly, however, we demonstrate that after CD3/CD28 engagement, deficiency of PKC (cid:2) primarily abrogates NFAT transactivation. In contrast, NF- (cid:4) B activation was only partially reduced. This NFAT transactivation defect appears to be secondary to reduced inositol 1,4,5-trisphosphate generation and intracellular Ca 2 (cid:3) mobilization. Our finding suggests that PKC (cid:2) plays a critical and nonredundant role in T cell receptor–induced NFAT activation. system as previously described (22). Nanomolar values of cytoplasmic Ca 2 (cid:3) concentration were calculated from the ratios of background subtracted images (excitation wavelength 350 and 385 nm, emis-sion wavelength 510 nm) according to the calibration procedure and equations previously described (23). Experiments have been performed at least four times with similar outcomes. Inositol 1,4,5-Trisphosphate (IP 3 ) Measurement. Mature CD3 (cid:3) T cells were stimulated by cross-linking of CD3 as described above. The stimulations were terminated by the addition of ice cold TCA followed by 15 min of incubation on ice. The samples were centrifuged at 1,400 g at 4 (cid:19) C for 15 min, and the supernatant was extracted with 10 vol water-saturated diethyl ether neutralized with 1 M NaHCO 3 . IP 3 was quantitated in duplicate samples using a competitive [ 3 H]IP 3 binding assay (Amersham Biosciences) according to the manufacturer’s instructions. Experiments have been performed at least three times in duplicates with similar outcomes.