A matched pair of tumorigenic and nontumorigenic human hybrids between GM0097 diploid fibroblasts and HT1080 fibrosarcoma cells was used to investigate further the relationship between the amount of surface fibronectin and the malignant potential of the cell. We can confirm that the malignant phenotype in these hybrids cosegregates with a decrease in cell-surface fibronectin. Moreover, the nontumorigenic hybrid incorporates into its extracellular matrix a significantly higher proportion of fibronectin isoforms that do not contain the alternatively spliced regions ED-A and ED-B. When, after chromosome segregation, malignant potential reappears in the hybrid cells, they incorporate more ED-A+ and ED-B+ isoforms than do the nontumorigenic hybrids from which they were derived.