Transforming growth factor-beta (TGF beta) stimulates bone formation in vivo and in vitro, related in part to an increase in type I collagen production. In osteoblast-enriched cultures from fetal rat bone, 24- to 48-h TGF beta 1 treatment enhanced collagen synthesis rates by 2.5- to 6-fold, while it increased collagen accumulation by 5- to 10-fold. These effects were not accounted for by similar changes in acid-soluble radioisotope, cell number, or steady state type I procollagen transcripts. Basal collagen synthesis and accumulation were markedly reduced when mRNA transcription was blocked with alpha-amanitin, but the relative stimulatory effects of TGF beta 1 persisted in toxin-treated cultures. Newly synthesized collagen was rapidly secreted into the culture medium. While pulse-chase studies demonstrated that total (medium plus cell-associated) collagen levels were stable throughout the 48-h period, TGF beta 1 increased the fraction of cell-associated collagen between 24-48 h, and this was partially blocked by alpha-amanitin, but not by antibody to fibronectin or beta 1-integrin subunit. TGF beta 1, therefore, has multiple effects on type I collagen in fetal bone-derived cell cultures, including small increases in mRNA, large increases in polypeptide synthesis, and enhanced association of secreted collagen to the cell layer, which may require synthesis of extracellular components unrelated to fibronectin or the beta 1-integrin subunit.