Chloramphenicol 3-O-phosphotransferase (CPT) from Streptomyces venezuelae ISP5230, a novel chloramphenicol-inactivating kinase, has been overexpressed and purified using Escherichia coli as the heterologous host. Crystals of CPT in complex with its substrate chloramphenicol (Cm) were obtained which were suitable for X-ray diffraction. The crystals belong to the cubic space group I4132 with unit-cell dimension a = 200.0 A. The initial CPT crystals diffracted to 3.5 A and the diffraction was improved significantly upon adding acetonitrile and Cm to the crystallization drop. The CPT-Cm crystals diffract to at least 2.8 A resolution.