Reversed-phase liquid chromatography was used as an alternative method for the characterization of the precursor and activated forms of porcine and human pancreatic colipase. Using a Beckman Ultrasphere column with an increasing acetonitrile gradient in 0.1% trifluoroacetic acid, it was possible to obtain well-resolved separation of the precursor form of colipase (procolipase) from its trypsin-activated derivative. This protocol was used (1) to study the activation of porcine procolipase by trypsin or thrombin in vitro, (2) to assess the homogeneity of porcine colipase preparations used in tridimensional structure studies and in combination with immunoaffinity chromatography, (3) to identify the form of colipase present in samples of human pancreatic juice.