We demonstrate that the alternatively spliced variable (V) region of fibronectin (FN) is required for secretion of FN dimers during biosynthesis. Alternative splicing of the V segment of the rat FN transcript generates three subunit variants (V120, V95, V0) that differ by the inclusion or omission of an additional 120 or 95 amino acids. We are exploring the functions of this segment by expressing variant cDNAs in normal and transformed fibroblasts. Like FN itself, the cDNA-encoded polypeptides (deminectins [DNs]) containing the V120 or V95 segment are efficiently secreted as disulfide-bonded homodimers. However, few homodimers of DNs lacking this region, V0 DNs, are secreted. V0 homodimers do form inside the cell, as demonstrated by biosynthetic analyses of dimer formation and secretion using pulse-chase and time course experiments, but these dimers seldom reach the cell surface and are probably degraded intracellularly. Coexpression of V0 and V120 subunits results in intracellular formation of three types of dimers, V0-V0, V0-V120, and V120-V120, but only the V120-containing dimers are secreted. This selective retention of V0 homodimers indicates that the V region is required for formation and secretion of native FN dimers. In an analogous in vivo situation, we show that plasma FN also lacks V0-V0 dimers and consists of V0-V+ and V+-V+ combinations. Dissection of V region sequences by deletion mapping localizes the major site involved in DN dimer secretion to an 18-amino acid segment within V95. In addition, high levels of dimer secretion can be restored by insertion of V into a heterologous site 10 kD COOH terminal to its normal location. We discuss the potential role of intracellular protein-protein interactions in FN dimer formation.