PKCβ was identified by gene expression profiling and confirmed by independent analysis as a possible rational therapeutic target for DLBCL. Therefore, we investigated the safety and anti-cancer activity of enzastaurin, an orally administered, potent inhibitor of PKCβ, in this disease. Patients (pts) with relapsed DLBCL, who progressed following CHOP-based (or equivalent) chemotherapy, were enrolled in this phase II, single-arm, US multicenter study. The primary objective was to determine the rate of freedom from progression ≥2 cycles (FFP). Secondary end-points included objective response rate (complete or partial response), and toxicity. Pts initially received an oral dose of 525 mg (capsules) enzastaurin, amended to 500 mg (tablets), once daily, until disease progression or unacceptable toxicity occurred. One cycle of therapy lasted for 28 days. All 55 enrolled pts received at least 1 dose of enzastaurin, and were included in the safety and efficacy analyses. Of these, 13 pts completed less than 1 cycle of therapy, which is noteworthy because enzastaurin must be administered for 14 days to achieve therapeutic levels. Of the 55 pts evaluated, 27 were men and 28 were women, 47 (85%) had an ECOG performance status of ≤1, and 28 pts had elevated LDH levels. The median age was 68 years (range: 31–87). Pts had received a median number of 2 prior therapies (range: 1–5); 6 pts had also received prior stem-cell transplantation. There were 11 dose omissions due to toxicities, 2 of which were possibly related to the study drug (fatigue and edema). There was only 1 grade 4 toxicity (hypomagnesemia), 1 grade 3 thrombocytopenia, and no grade 3 or 4 neutropenia. Other grade 3 toxicities were fatigue (2), edema (1), headache (1), and motor neuropathy (1). Twelve of 55 pts (21.8%, 95% CI: 11.8%–35.0%) were alive and free from progression at the end of 2 cycles. Two of these 12 pts had elevated LDH levels at baseline, which normalized after enzastaurin therapy. Nine pts had stable disease for at least 4 cycles, one of whom achieved a complete response; 2 of the 9 pts had relapsed after prior stem-cell therapy. Five pts with stable disease achieved a minor response (lesion shrinkage ≥25% and In conclusion, several pts with multiple relapsed DLBCL achieved prolonged periods of stable disease following enzastaurin treatment, although the objective tumor response rate was low. In addition, enzastaurin was well tolerated. These results suggest that enzastaurin, an oral agent, has clinical activity in DLBCL that warrants further investigation.