Recent reports have described transformation- and tumour-specific expression of fibronectin isoforms generated by alternative splicing of the fibronectin pre-mRNA. We have investigated the expression and distribution of EDIIIA+ and EDIIIB+ fibronectin splice variants in tumorigenic and non-tumorigenic somatic cell hybrids made by fusing fibrosarcoma-derived cells (HT1080) and normal fibroblasts (GM00097). Alternative splicing of EDIIIA and EDIIIB was assessed quantitatively by S1 nuclease analyses. The levels of EDIIIA+ and EDIIIB+ fibronectin mRNAs were similar in the parental and hybrid cells. Domain-specific monoclonal antibodies were used in immunohistochemical studies to identify EDIIIA+ and EDIIIB+ fibronectins in fixed cells. GM00097 and the non-tumorigenic hybrid (clone G3) showed high levels of both EDIIIA+ and EDIIIB+ fibronectin staining. The tumorigenic hybrid (clone C1) showed reduced amounts of EDIIIA+ fibronectin, but no detectable EDIIIB+ fibronectin. No fibronectin was detected on the surface of HT1080 cells. Western blots of protein extracted from culture supernatants and extracellular matrices revealed that GM00097 and G3 cells incorporated most of the EDIIIA+ and EDIIIB+ fibronectin into the extracellular matrix whereas C1 cells released a large proportion of the EDIIIA+ fibronectin, and almost all of the EDIIIB+ fibronectin, into the supernatant. We conclude that there are differences in the presence of EDIIIA+ and EDIIIB+ FNs on the surface of tumorigenic and non-tumorigenic cells and that these differences are due to differential incorporation of FN variants into the ECM.