Multiple fibronectin mRNAs arise by alternative splicing of the primary transcript of a single gene. We describe analyses of the contribution of this alternative splicing to fibronectin subunit heterogeneity in three different cell types using antisera directed against specific segments of fibronectin. beta-galactosidase-fibronectin fusion proteins produced with the lambda gt11 bacterial expression vector were used as immunogens. One region of alternative splicing accounts for differences in subunit size, while a second contributes to differences between the fibronectins present in blood plasma and in fibroblastic cells. We also show, however, that these two regions of alternative splicing do not account for all detectable subunits. We have also used these segment-specific antisera to show that blood platelets contain a spectrum of fibronectin subunits distinct from that found in blood plasma.