The growth-promoting activities of tumor-conditioned media (TU-CM) obtained from 23 cultured human nonfunctioning pituitary adenomas were studied in vitro. TU-CM obtained from adenoma cell cultures increased both cell counts (range: 108-179%; control = growth in serum-free medium = 100%) and 3H-thymidine incorporation (112-139%) of rat pituitary cell cultures, indicating that TU-CM contains growth-stimulating substances. TU-CM was also able to stimulate the growth of normal fibroblasts (3H-thymidine incorporation: 164-178%; cell counts: 145-157%) and endothelial cells (3H-thymidine incorporation: 131-149%; cell counts: 181-217%), suggesting the presence of - possibly angiogenic-growth factors that act on these cell types. However, the growth of hormone-producing cells was also stimulated, since TU-CM increased 3H-thymidine incorporation into rat pituitary cells in the presence of D-Val-MEM, a medium specifically inhibiting growth of fibroblasts. Addition of neutralizing antibodies against transforming growth factor alpha (TGF-alpha), epidermal growth factor (EGF), insulin-like growth factor I (IGF-I) and basic fibroblast growth factor (bFGF), either alone or in different combinations, reduced the growth-promoting activity of TU-CM on rat pituitary cells (range: 96-71%; control = growth effect of TU-CM without antibodies = 100%), strongly indicating the presence of these growth factors in TU-CM. All 4 antibodies together completely inhibited the growth-stimulatory activity of TU-CM, strongly suggesting that these growth factors play the major role among growth-stimulating substances in TU-CM. This is the first study giving evidence that TGF-alpha, EGF, IGF-I and bFGF are secreted by nonfunctioning adenoma cells indicating that the growth factors could be involved in growth regulation of pituitary adenomas by paracrine or autocrine mechanisms.