Type β transforming growth factor (TGFβ) was shown to regulate the production of several extracellular matrix proteins. Osteopontin (OP) is a recently discovered bone matrix protein which was shown to promote the attachment of osteoblastic rat osteosarcoma ROS 17/2.8 cells to their substrate. We examined the effects of TGFβ on OP production and OP mRNA in ROS 17/2.8 cells. Four-day treatment with 4 ng/ml TGFβ1 increased substantially the level of osteopontin in the cell culture media, as estimated by immunoblotting. Metabolic labeling showed that this effect was associated with a 3-4-fold increase in OP biosynthesis. TGFβ1 also increased, in a dose-dependent manner starting at 0.4 ng/ml, the steady-state level of OP mRNA. The increase in OP mRNA was first detected 48 h after the addition of TGFβ1 and lasted at least until 120 h. The half-life of OP mRNA, estimated in the presence of 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, was about 10 h and was not altered by TGFβ1. On the other hand, the increase in OP mRNA was blocked by actinomycin D. Nuclear run-on assays indicated that TGFβ1 increased the rate of transcription of the OP gene. Examination of hormonal interactions showed that TGFβ1 opposed or compensated for the reduction in OP mRNA produced by dexamethasone and that TGFβ1 did not further augment OP mRNA levels which had been increased by 1,25-dihydroxyvitamin D3 treatment. TGFβ2 had similar effects on OP gene expression as TGFβ1. In conclusion, TGFβ promotes the production of osteopontin in the osteoblastic osteosarcoma cells through a pathway which is at least in part mediated by transcriptional events.