Abstract Four pregnant cows near the end of lactation were fitted with a catheter in a lymph duct afferent to the supramammary lymph node. Cows were dried off 3 d after surgery, and samples of lymph were collected daily from the day of surgery until 4 d postpartum. Samples of blood and mammary secretions were taken before and after drying off and at parturition. Concentrations of most metabolites were lower in lymph than in serum. Concentrations of IGF-I and prostaglandin E2 were not affected at drying off but decreased and increased, respectively, at parturition. All IGF-binding proteins that were present in serum were also present in lymph fluid, but the binding activity was lower. Mitogenic activities of lymph samples taken at various physiological stages were determined on mammary epithelial (MAC-T) and fibroblast cell lines. Lymph was mitogenic, but mitogenic activity was not related to physiological stages. The correlation was high between mitogenic activity of lymph on MAC-T cells and the content of prostaglandin E2 in lymph. Supplementation of lymph with additional prostaglandin E2 increased mitogenic activity, and neutralization of lymph by antibodies reduced mitogenic activity. Basal medium conditioned by the epithelial cell line contained 100 to 250 pg/ml of immunoassayable prostaglandin E2.