Activation of PKC contributes to tumor cell survival and proliferation and has been implicated in the pathogenesis of human B cell lymphomas. Specifically, PKCβ activation is increased in tumor cells from patients with poor prognosis diffuse large cell lymphoma (DLCL), suggesting that PKCβ may be a target for therapeutic intervention. In this study we tested the PKCβ inhibitor, Enzastaurin (LY317615), using both cell culture and xenograft assays, on a panel of eight DLCL lines (OCI-Ly3, OCI-Ly7, OCI-Ly10, OCI-Ly19, SUDHL-4, SUDHL-6, Farage, and Toledo). In suspension culture, Enzastaurin was cytotoxic to all lines tested, with an average IC50 at 48 hours of 2.0 micromolar (range 0.5–4.0 micromolar). Molecular analyses showed that Enzastaurin treatment inhibited phosphorylation of GSK3β, a potential pharmacodynamic marker of Enzastaurin activity. Subsequent studies were performed using two subclones of the DLCL lines OCI-Ly10 and OCI-ly19, which respectively represent the ABC (activated B cell) and GC (germinal center) molecular subclasses of DLCL. Each line was engineered to express luciferase, which permits real time in vivo imaging. Upon subcutaneous transplantation into immune deficient NOD/SCID mice, both lines formed reproducible tumors. Treatment with Enzastaurin (75mg/kg, b.i.d. oral gavage) was initiated during the first 1–2 weeks post-transplant, before palpable tumors were evident, and was continued for 14 consecutive days. The Enzastaurin treatment was well tolerated, with the only overt side effect being moderate weight loss. At the end of therapy, vehicle control treated tumors had become palpable and progressed to volumes of 40–60 cubic millimeters. In contrast, Enzastaurin treated tumors (both OCI-Ly10 and OCI-Ly19) showed no observable progression, remaining detectable by luciferase imaging, but non-palpable. Collectively, these data indicate the Enzastaurin can induce death of human DLCL lines in vitro and is at least cytostatic to human DLCL in vivo.