Imatinib (Gleevec) is a multiple tyrosine kinase inhibitor that decreases the activity of the fusion oncogene called BCR-ABL (breakpoint cluster region protein-Abelson murine leukemia viral oncogene homolog) and is clinically used for the treatment of chronic myelogenous leukemia and acute lymphocytic leukemia. Small molecule drugs, such as imatinib, can bind to several cellular proteins including the target proteins in the cells, inducing undesirable effects along with the effects against the disease. In this study, we report the synthetic optimization for 14 C-labeling and radiosynthesis of [14 C]imatinib to analyze binding with cellular proteins using accelerator mass spectroscopy. 14 C-labeling of imatinib was performed by the synthesis of 14 C-labeld 2-aminopyrimidine intermediate using [14 C]guanidine·HCl, which includes an in situ reduction of an inseparable byproduct for easy purification by HPLC, followed by a cross-coupling reaction with aryl bromide precursor. The radiosynthesis of [14 C]imatinib (specific activity, 631 MBq/mmol; radiochemical purity, 99.6%) was achieved in six steps with a total chemical yield of 29.2%.