The aim of this study was to determine the principal active molecules in the acid phosphatase (AcP) fraction of skim milk origin using immunostaining and AcP staining. The AcP fraction was separated from skim milk at 0.38 m NaCl using carboxymethyl cellulose chromatography at pH 5.2. The molecular mass of the active molecule in AcP fraction was estimated to be 80 kDa by immunostaining and AcP staining. The 80 kDa protein was analyzed by a protein sequencer, using the automated Edman degradation method; the first thirteen N-terminal amino acid sequence obtained were shown to be APRKNVRWXTIXQ. For that amino acid sequence, there was 84% (11/13 residues) homology with the amino acid sequence of bovine lactoferrin (LF). The AcP fraction and commercial LF showed a similar AcP activity profile, having an optimum pH of 4.5 and temperature of 60 degrees C. Thus, the AcP fraction from bovine skim milk was isolated and the principal active molecule present was tentatively identified as LF (C) 2009 Elsevier Ltd. All rights reserved.