Pantothenate-p-nitroanilide has been synthesized for use as a substrate in a continuous spectrophotometric assay of pantetheinase activity monitoring absorbance at 41 0 nm. Pantothenate-p-nitroanilide is a crystalline compound with a molecular weight of 338.0 and a melting point of 146-149°C. Use of this substrate in the described assay is suitable for enzyme activity determination in high protein content media such as blood serum. Serum pantetheinase activity was determined for rats of varying pantothenate nutriture. Rats with mildly (but significantly, p