The amino- and carboxyl-terminal globular domains of type VI collagen are composed of several homologous modules similar to the type A collagen-binding modules present in von Willebrand factor. The human alpha 3(VI) chain that contributes most of the amino-terminal globule appears heterogeneous in size as a result of alternative splicing of two exons (Stokes D. G., Saitta, B., Timpl, R., and Chu, M.-L. (1991) J. Biol. Chem. 266, 8626-8633). In the present study, we report a further characterization of the 5'-end of the gene of the human alpha 3(VI) chain and show that transcription initiates at multiple sites. Southern blotting and DNA sequencing indicate that there is an additional type A exon (A9/N10) at about 1.8 kilobase pairs downstream of the exon coding for the signal peptide. The open reading frame of this additional exon reveals 1 cysteine and three potential N-glycosylation sites. Polymerase chain reaction, Northern blotting, and RNase protection assays demonstrate that exon A9/N10 is subject to alternative splicing in normal and tumor cell lines and that this generates more protein variants of the alpha 3(VI) chain than expected before. A comparison with the corresponding amino-terminal globule of the chicken alpha 3(VI) chain shows the presence of 1 additional cysteine in this portion of the molecule and suggests that human type VI collagen has more possibilities for structural and functional variations compared to chicken type VI collagen.