Pantothate kinase (E.C.2.7.1.33) was partially purified from rat liver and analyzed by preparative isoelectric focusing and cation-exchange high performance liquid chromatography. Two main peaks of catalytic activity were found in the focusing gel at isoelectric point of 5.7 (peak A) and 5.1 (peak B). Two major molecular forms of the enzyme was also demonstrated by high performance liquid chromatography. Both molecular forms of the enzyme were inhibited by 0.02 mmol/l CoASH and acetyl-CoA. CoA at 0.02 mmol/l inhibited the isoenzyme peak B only slightly (6%) compared to the isoenzyme peak A (80%). Acetyl-CoA at 0.02 mmol/l inhibited peak A and B with about equal strength (96% and 83% respectively). The relative amounts of these two molecular forms of pantothenate kinase in different organs might contribute to the regulation of CoA synthesis in mammalian cells.