Here we describe the evaluation of 3'-[(18)F]fluoro-3'-deoxythymidine [[(18)F]-FLT] as a tracer for positron emission tomography (PET) in a murine model of B-cell lymphoma and in human malignant lymphoma. The human B-cell line DoHH2 expressed high levels of active thymidine kinase 1 (TK-1) as the key enzyme of [(18)F]-FLT metabolism. Immunostaining confirmed high levels of TK-1 in DoHH2 derived xenograft tumors in SCID/SCID mice. In vitro studies demonstrated a time-dependent uptake of [(18)F]-FLT, an efficient phosphorylation to the respective monophosphate and the incorporation of [(18)F]-FLT into the perchloric acid insoluble fraction in DoHH2 cells, indicating the incorporation of this tracer into the DNA. After incubation with [(18)F]FLT for 240 min, 12.5% +/- 1.0% of radioactivity applied to the medium was intracellularly trapped in DoHH2 cells. Specific accumulation of [(18)F]-FLT in the malignant cell clone was confirmed in biodistribution studies in SCID/SCID mice bearing DoHH2-derived tumors. The percentage of injected dose of [(18)F]-FLT per gram of tumor tissue correlated with the tumor-proliferation index as evaluated in BrdUrd-labeling experiments. In a pilot study of 11 patients with both indolent and aggressive lymphoma, [(18)F]-FLT was suitable and comparable to [(18)F]-FDG in the ability to detect malignant lesions by PET scan. Furthermore, we found a close correlation (r = 0.95, P < 0.005) of the [(18)F]-FLT standardized uptake values with the Ki67-labeling index of tissue biopsies (n = 10) in these patients. These results suggest that [(18)F]-FLT represents a novel tracer for PET that enables imaging of proliferation in human lymphoma in vivo.