The synthesis and secretion of fibronectin (FN) was observed in 4 human hepatocellular carcinoma (HCC)-derived cell lines using a serum-free conditioned medium. It was also demonstrated that FNs secreted from these cell lines (HCC-FN) were structurally altered compared to plasma FN produced by normal hepatocytes. Using limited proteolysis with cathepsin D followed by immunoblot analysis with a carboxy-terminal specific antibody, the existence of an extra domain (ED) segment in the carboxy-terminal region of HCC-FNs was confirmed. Expression of the additional nucleotide insert (ED sequence), which is reported to be absent in hepatocyte mRNA and is characteristic of cellular FN-mRNA, was confirmed in mRNA from all 4 HCC cell lines. It is now evident that HCC cells secrete different variants of FN compared with normal hepatocytes due to alternative splicing of mRNA precursors. This differential RNA processing seems to be one of the phenotypic alterations associated with the oncogenic transformation of hepatocytes.