Studies on osteosarcoma cell lines point to the potential importance of transforming growth factor beta (TGF beta) as an autocrine factor which controls the growth of human osteosarcomas. To define further the role of TGF beta isoforms in these neoplasms, a series of 27 osteosarcomas was studied using immunohistochemical, mRNA in situ hybridization, and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. All 14 central high-grade osteosarcomas, two telangiectatic osteosarcomas, and one high-grade surface osteosarcoma showed cytoplasmic immunoreactivity for TGF beta 1, -2, and -3. The expression of TGF beta 1 was moderate or diffuse in 14 cases (82.3 per cent), while low expression was detected in only three cases (17.7 per cent). For TGF beta 2 and -3, only moderate or diffuse staining was observed. Low-grade parosteal and periosteal osteosarcomas showed low or undetectable levels of TGF beta 1, while TGF beta 2 and -3 were moderately or diffusely expressed. Finally, three dedifferentiated parosteal osteosarcomas were diffusely positive for TGF beta 1, -2, and -3 in the high-grade component, while in the low-grade component, available for analysis in two of these cases, TGF beta 1 was demonstrated in a few neoplastic cells, and TGF beta 2 and -3 maintained a diffuse distribution. Statistical analysis of these data showed that high-grade osteosarcomas had a significantly higher expression of TGF beta 1 than low-grade osteosarcomas, while levels of TGF beta 2 and -3 were comparable in the two groups (p < 0.001; p = 0.3; p = 0.3, respectively; Fisher's exact test). Similarly, mRNA levels of TGF beta 1 detected by in situ hybridization were significantly higher (p = 0.04, Fisher's exact test) in high-grade osteosarcoma variants, while no differences were found for TGF beta 2 and -3 mRNA (p = 1.0; p = 0.2, respectively; Fisher's exact test). In addition, mRNA analysis performed by RT-PCR in seven cases (five high-grade and two low-grade osteosarcomas) confirmed the presence of high levels of TGF beta 1 in high-grade osteosarcomas, while low-grade tumours had low or absent mRNA expression. In conclusion, this positive association suggests that TGF beta 1 may be involved in determining the aggressive clinical behaviour of high-grade osteosarcomas.