IGF-I and -II are peptide growth factors that may be important contributors to the growth-promoting properties associated with milk. IGF in extracellular fluids, including serum and milk, are carried by specific high-affinity binding proteins (IGFBP). In this study, the levels of IGF-I and -II in rat serum and milk were quantified by specific RIA, and the IGFBP were characterized using Western ligand blotting and autoradiography throughout lactation. The levels of IGF-I in both milk and maternal serum decreased during lactation. Serum IGF-I decreased from 743 ≤ 187 μg/L on d 1 to 391 ≤ 106 (mean ≤ SD) on d 21 of lactation, and milk IGF-I levels fell from 30 ≤ 10 to 13 ≤ 8 μg/L. Levels of IGF-II in serum and milk were much lower than IGF-I, and were unaffected by lactation. In maternal serum, several IGFBP were identified: IGFBP-3, which migrates as four glycosylated bands with apparent M, from 38 to 42 kD and one to two nonglycosylated bands with apparent M, of 28 to 29 kD, and an IGFBP with an apparent M, of 24 kD. In milk, IGFBP-3, the 24-kD binding protein, and a third IGFBP with an apparent M, of 29 kD were identified. Treatment of milk and serum with Endoglycosidase F reduced the four glycosylated IGFBP-3 bands (38–42 kD) to two bands with apparent M, of 35 and 32 kD. In rat milk, but not adult rat serum, the IGFBP with an apparent M, of 29 kD was immunoprecipitable by an antibody that recognizes IGFBP-2. These results demonstrate that in adult rat serum and milk both IGF-I and IGF-II are present, with IGF-I being predominant. IGFBP are present in rat milk; the presence of IGFBP-2 in rat milk, but not in maternal serum, indicates that IGFBP may be synthesized within the mammary gland.