Abstract: We have synthesized several new chromogenic substrates, p-nitroanilides of the dipeptides, Gly-Pro, Ala-Pro, Lys-Pro, Arg-Pro, Glu-Pro, and Asp-Pro, for X-prolyl dipeptidyl-aminopeptidase. These have permitted the development of a simple assay of the enzyme in which p-nitroaniline liberated directly or after the Bratton-Marshall reaction is measured spectrophotometrically. The enzyme activity was measured in human serum or in homogeneous enzyme purified from human submaxillary gland. The homogeneous enzyme hydrolyzed each substrate to produce X-Pro and p-nitroaniline. The optimum pH was at 8.7, except with Arg-Pro p-nitroanilide (8.0). Serum enzyme hydrolyzed Gly-Pro p-nitroanilide to p-nitroaniline and Gly-Pro, which was further hydrolyzed to Gly and Pro by an imidodipeptidase in serum. Gly-Pro β-naphthylamide or Gly-Pro-Leu was a competitive inhibitor with each X-Pro p-nitroanilide as substrate. Gly-Pro p-nitroanilide had the highest activity among the substrates at pH 8.7, followed by p-nitroanilides of Ala, Lys, Arg, Glu, and Asp in a decreasing order of activity.