A biologically active, low-molecular-weight, chromium-binding substance present in milk (M-LMCr) was isolated from bovine colostrum and purified more than 2000 times by means of ethanol precipitation and successive ion-exchange and Sephadex gel chromatographies. The purified M-LMCr appeared to be an anionic organic Cr compound with a molecular weight of 1500, as determined by gel permeation chromatography. It contained aspartic acid, glutamic acid, glycine and cysteine in a ratio of 5:4:2:1 and no detectable carbohydrate. Although we were unable to detect nicotinic acid, some ultraviolet-absorbing (lambda max 260 nm) chemical structure was shown to be a constituent. Purified M-LMCr stimulated the rates of both [U-14C]glucose oxidation and [3-3H]glucose conversion into lipid in rat epididymal adipocytes at Cr concentrations greater than 1.5 ng/mL in relation to insulin action. This substance appears to have properties similar to those of glucose tolerance factor in yeast and the low-molecular-weight, chromium-binding substance present in mammalian liver. The role of M-LMCr in Cr nutrition and detoxication is discussed.