The production of transforming growth factor-beta 2 (TGF-beta 2), interleukin-1 beta (IL-1 beta), IL-6, IL-8, and tumor necrosis factor-alpha (TNF-alpha) by spontaneously immortalized human mammary gland epithelial cells of non-malignant origin and the effect of prolactin upon the production of those cytokines were investigated. Cells were cultured on plastic with epithelial growth factor, insulin, and hydrocortisone. Cytokines were quantified by enzyme-immunoassays. The cells produced IL-6 and IL-8, but no detectable TGF-beta 2, IL-1 beta, or TNF-alpha. Although prolactin enhanced the uptake of [3H]thymidine, it did not alter the production of cytokines/interleukins. Because of the marked production of IL-8 by mammary epithelium and a past report of TGF activity in human milk, those agents were quantified in human milk. The mean +/- S.D. concentrations of IL-8 and TGF-beta 2 in human milk obtained in the first 3 days of lactation were 3684 +/- 2910 and 130 +/- 108 pg/ml, respectively. Thus, IL-8 and TGF-beta 2 are normal constituents in human milk, and human mammary gland epithelium may be responsible for producing some of the IL-6 and IL-8 in human milk.