The mitogenic activity of milk from different stages of lactation was studied in primary cultures of undifferentiated bovine mammary epithelial cells. The mitogenic activity, measured as DNA synthesis, was 3-4-fold higher in colostrum than in basal medium. The mitogenic activity declined rapidly after calving, and in mid and late lactation the effect was inhibitory, not stimulatory. The content of IGF-I in milk varied with stage of lactation declining from more than 300 ng per mi in colostrum to 1-2 ng per ml in mid lactation and increasing to 20 ng per mi in late lactation. The difference in mitogenic activities between colostrum and mature milk was closely related to the difference in IGF-I content. The changes in IGF-I, however, cannot account for the inhibitory effect of mid and late lactation milk and the difference in the mitogenic effect of colostrum and BPMS (bovine prepartum milk-like secretion). The amounts of IGF binding proteins (24, 28, 34 and 41-44 kDa) in milk followed the same pattern as IGF-I with high content in early lactation and low content in mid lactation. The discrepancies between mitogenic activity and IGF-I content may in some, but not all, cases be related to the content of binding proteins. (C) 2001 Elsevier Science B.V. All rights reserved.