We describe here a third region of variability in human fibronectin (FN) due to alternative RNA splicing. Two other positions of alternative splicing have been reported previously (ED and IIICS). The third region involves a 273-nucleotide exon encoding exactly one 91-amino acid repeat of type III homology, located between the DNA- and the cell-binding domains of FN, which is either included in or excluded from FN mRNA. The two mRNA variants arising by an exon-skipping mechanism are present in cells known to synthesize the cellular form of FN. However, liver cells, which are the source of plasma FN, produce only messengers without the extra type III sequence. Therefore, the region described here resembles, both structurally and functionally, the previously described ED (for extra domain) region, located toward the C terminus of the molecule, between the cell- and heparin- (hep 2) binding domains. We conclude that both the extra type III repeat (named EDII) and ED represent sequences restricted to cellular FN. Combination of all the possible patterns of splicing in the three regions described to date may generate up to 20 distinct FN polypeptides from a single gene.