Bovine mammary gland secretions from several developmental stages were shown to stimulate [3H]thymidine incorporation into AKR-2B mouse embryo fibroblast cells. In virgin heifers, mammary secretions at 1% concentration stimulated thymidine incorporation into AKR-2B cells more than threefold compared with 10% (v/v) fetal calf serum. The growth-promoting activity peaked at an early stage of the last trimester (7-8 months) of gestation and declined after this until the colostrum forming stage. At this point, the activity was one- to twofold that induced by 10% (v/v) fetal calf serum. At parturition the activity dropped abruptly to virtually undetectable values in milk. The developmental change in activity could be mimicked by hormonal priming of the mammary gland. The partial characterization of the growth promoting activities in secretions at the seventh month of gestation revealed at least two major growth-promoting activities: bovine mammary derived growth factor 1 (bMDGF-1), an epidermal growth factor-like growth factor with a molecular weight of 30,000, which is trypsin sensitive and heat stable, and bMDGF-2, which eluted under gel filtration conditions at a molecular weight of 50,000 and 150,000. bMDGF-1 is predominant in pregnant, precolostric, and colostric secretions, and is not detected in milk. bMDGF-2 is the major growth factor in milk. These results show developmental regulation and modulation of growth-promoting factors during the different stages of mammary gland development and suggest that growth factors are involved in regulating growth during gestation.