The 64CuCu-PTSM radiopharmaceutical, pyruvaldehyde bis(N4-methylthiosemicarbazonato)copper(II), is suitable for use in microPET and autoradiographic imaging to assess regional tissue perfusion in small animal models. We report here an approach to synthesis and formulation of the 64CuCu-PTSM radiopharmaceutical at the high concentrations required for use in imaging with rodent models of human disease. The 64CuCu-PTSM radiopharmaceutical was prepared at small volumes by addition of the H2PTSM ligand to acetate-buffered 64Cucopper chloride, followed by solid phase extraction to isolate and purify the product, which was then recovered and formulated in 2-mL normal saline containing 5% ethanol and 5% propylene glycol. Results. The 64CuCu-PTSM radiopharmaceutical has been produced over the range of 0.41–1.85 GBq (11–50 mCi) 64CuCu-PTSM in the 2.0-mL final product volume. Radiochemical purity of the 64CuCu-PTSM radiopharmaceutical product averaged 99.8 ± 0.4% (n = 64), with the final formulated product produced at an 83 ± 5% radiochemical yield. The approach to 64CuCu-PTSM synthesis and formulation has proven to be reliable and robust, supporting radiopharmaceutical delivery at the high concentrations required for PET studies in mouse and other rodent models. Display omitted •64CuCu-PTSM prepared in a formulation suitable for small rodent models.•Rapid and reliable synthesis producing radiopharmaceutical in range of 0.2–0.9 GBq/mL.•64CuCu-PTSM product delivered with high radiochemical purity (99.8 ± 0.4%; n = 64).•Allows microPET or autoradiographic imaging to map regional blood flow.