Erbstatin, a specific inhibitor of tyrosine protein kinase isolated from Streptomyces sp. MH435-hF3, exhibits temporary effects in cell culture, likely due to its short half-life in the medium. To confirm this possibility, we biosynthetically prepared radioisotope-labeled erbstatin using the erbstatin-producing strain and studied its stability in culture medium. We tested L-[U-14C]tyrosine, L-[U-14C]phenylalanine, and [G-14C]shikimic acid as precursors, optimized medium conditions (fish meal concentration and a synthetic medium) to enhance specific activity, and analyzed the stability of erbstatin in serum-free DMEM and its accumulation in A431 cells. Results showed tyrosine was the most effective precursor for labeling erbstatin; [3H]erbstatin prepared from the synthetic medium had a 110-fold higher specific activity than that from the fish meal-containing medium. Erbstatin in serum-free DMEM had a half-life of approximately 20 minutes, with only 13% remaining after 2 hours. Intracellular erbstatin in A431 cells peaked at around 30 minutes and decreased to about 45% of the peak amount by 3 hours. The labeled compound should be useful for further studies on the subcellular distribution of erbstatin.