AbstractTwo different mitogenic activities were identified from extracts of porcine pituitary by using COMMA‐D mouse mammary epithelial cells in a serum‐free 3H‐thymidine incorporation assay. Porcine pituitaries were extracted in phosphate‐buffered saline (pH 7.4) and 25–80% (NH4)2SO4 pellets were dialyzed and chromatographed by using DEAE‐Sepharose Chromatography (pH 8.0), resulting in two peaks (I and II) of mitogenic activity. Peak I represented a recovery of 73% of the units of mitogenic activity present in crude extract of pituitary while only 1.25% of the activity was recovered in peak I was further purified by using CM‐Sephadex and heparin‐Sepharose chromatographies and yieled a mitogen that was able to elicit one‐half‐maximal stimulation of 3H‐thymidine incorporation by COMM‐D cells at 48 pg/ml. As expected with pituitary as the tissue source, peak I was confirmed to be basic fibroblast growth factor (bFGF) by using specific antibodies in enzyme‐linked immunosorbent assay and Western immunoblotting pocedures. Peak II was futher purified by using chromatorofocusing (pH 7.3–5.0), reverse‐phase, and action‐exchange HPLCs. The mitogenic activity eluted at pH 6.3 from chromatofocusing, migrated as a 13‐kDa molecule on gel filtration HPLC, and did not bind to heparin‐Sepharose under conditions which bound fibroblast growth factors. The material purified from peak II and rat synthetic transforming growth factor α (TGFα) competed in a parallel fashion with 125I‐epidermal growth factor for receptor binding with A431 human epidermal carcinoma cells. In addition, the mitogen purified from peak II showed a single immunoreactive band migrating at 15 kDa when specific antiserum against TGfα was used in a Western immunoblotting procedure. The data suggest that in addition to the well‐documented presence of bFGF, normal adult porcine pituitaries contain a 15‐kDa form of immunoreactive TGFα that binds to EGF receptors and is mitogenic for mammary epithelial cells.