Three fibronectin mRNAs exist in rat liver, differing by the presence or absence of segments of 285 or 360 bases at a point within the coding region. We previously proposed that the three mRNAs are encoded by a single gene and arise via alternative splicing of a common transcript. In order to test this hypothesis, we have isolated clones spanning approximately half of the fibronectin gene from a Fisher rat genomic library; blot hybridization analyses reveal the presence of only one fibronectin gene in the haploid rat genome. We determined the sequence of a portion (1221 nucleotides) of this gene. This sequence shows clearly that the three fibronectin mRNAs encoded by this gene are generated by a pattern of alternative splicing in which one 5' splice site can be paired with any one of three 3' splice sites, one at the beginning of, and two within, a single complex exon.