The transforming growth factor-β (TGFβ) family of mediators consists of five closely related isoforms, of which three are present in mammals. TGFβ₁ has been shown to exert a biphasic effect on the proliferation of several cell types, including fibroblasts, with stimulation at low concentrations and inhibition at higher concentrations. The stimulatory effects are well characterized, but the mechanisms by which TGFβ₁ inhibits cell proliferation are incompletely understood. In the present study we have compared the effects of all three mammalian TGFβ isoforms on human lung fibroblast proliferation, and have elucidated the role of the TGFβ-induced synthesis of prostaglandin E₂ (PGE₂) in mediating their actions. All three isoforms stimulated fibroblast proliferation with maximal effects at 5 pg/ml (0.2 pM) and an order of potency of TGFβ₃ > TGFβ₂ > TGFβ₁. At higher concentrations, proliferation declined, and at 40 pg/ml and above all isoforms inhibited fibroblast proliferation. Again TGFβ₃ was the most potent, but there were no significant differences between the inhibitory effects of TGFβ₁ and TGFβ₂. Addition of indomethacin, an inhibitor of PGE₂ synthesis, did not alter the proliferative activity of any of the TGFβ isoforms, but completely overcame their inhibitory effects, restoring the stimulatory actions observed at lower TGFβ concentrations. All TGFβ isoforms stimulated PGE₂ synthesis; TGFβ₃ was approximately twice as potent as TGFβ₁ and TGFβ₂, each of which had similar effects. These data suggest that the inhibition of fibroblast proliferation at higher concentrations of TGFβ isoforms may be mediated by autocrine stimulation of PGE₂ synthesis.