Lactoferrin (LF) is a main iron-transfering glycoprotein of human barrier body fluids, blood and milk. LF, a protein of the acute phase, is responsible for nonspecific cells defense against microbial and viral infection and cancer diseases. LF is an important component of the passive immunity of newborns system. LF, an extremely polyfunctional protein, is the object of intensive investigations. In this work electrophoretically homogeneous LF from human milk was prepared. Affinity chromatography of LF on Blue Sepharose separated the protein into several distinct isoforms with different affinities to this resin. Two of this isoforms possess nucleoside-5'-triphosphate-hydrolyzing activity. Using several methods including in-gel ATPase activity assays, we show that ATP (and others NTP) hydrolysis is an intrinsic property of LF, and that LF is the major ATPase of human milk. It was shown that ATP-hydrolyzing site is located in C-lobe of LF.