Multiple different subunits of fibronectin are known to occur and their origin has been unclear. Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing suggested an explanation for some of this diversity of fibronectin subunits. Because the alternative splicing events occur within the coding region, the mRNAs differ in coding potential. We have prepared recombinant phage containing a rat fibronectin cDNA segment that is present in some fibronectin mRNAs and not in others. This segment was inserted in the beta-galatosidase gene of lambda gt11, and fusion protein produced by lysogens of the recombinant phage was purified and used as immunogen. The resulting antisera recognized some subunits of rat and hamster fibronectins but not others, indicating that inclusion or removal of this segment gives rise to mRNAs that encode different fibronectin subunits. In particular, presence or absence of a 95 amino acid segment appears to account for differences in size among the subunits of plasma fibronectin, whose origin is therefore explained by alternative patterns of RNA splicing.