Human neonatal fibroblasts in monolayer culture produce insulin-like growth factor-binding protein-3 (IGFBP-3), the IGF-binding subunit of the circulating 140-kDa IGFBP complex. We now report that transforming growth factor-beta (TGF beta) is a potent stimulator of IGFBP-3 production by fibroblasts. After 72-h incubation with 1 ng/ml TGF beta, the levels of IGFBP-3 in conditioned medium were increased 5.8 +/- 1.2-fold (mean +/- SE; n = 9). Half-maximal stimulation of IGFBP-3 production was seen at 0.4 +/- 0.05 ng/ml TGF beta (n = 4). Coincubation of fibroblasts with TGF beta and either IGF-I or IGF-II at 50 ng/ml enhanced IGFBP-3 production 1.5- to 2-fold compared to TGF beta alone. As previously reported, fetal calf serum (FCS) stimulated IGFBP-3 production 5- to 6-fold; 1 ng/ml TGF beta increased the stimulated production of IGFBP-3 by FCS a further 2.5- to 3.5-fold. Acidification of FCS before addition enhanced the stimulation of IGFBP-3 compared to that caused by untreated FCS, but decreased further potentiation by TGF beta. This effect of acidified FCS was reversed by a neutralizing antibody to TGF beta. Similarly, the stimulation of IGFBP-3 levels by human serum or conditioned serum-free fibroblast medium was significantly increased by acidification of serum or medium before addition and was reversed by TGF beta antibody. These observations are consistent with acid-mediated activation of latent TGF beta added in serum or secreted by fibroblasts. Since IGFBP-3 is known to regulate IGF activity in fibroblasts, these results raise the possibility that TGF beta may modulate IGF actions in these cells by stimulating the production of IGFBP-3.