When collagen CNBr-cleavage peptides are analysed by two-dimensional gel electrophoresis each peptide is resolved into a reproducible set of charged forms. To test whether this peptide heterogeneity resulted from polymorphic mRNA, collagen was produced by transcription and translation in vitro of a collagen cDNA clone, and the peptides were mapped by two-dimensional gel electrophoresis. A cDNA construct was produced by ligation of the 5' end of the rat phenylalanine hydroxylase cDNA [Dahl & Mercer (1986) J. Biol. Chem. 261, 4148-4153], containing the translation-initiation codon, to a human alpha 1(I) cDNA [Chu, Myers, Bernard, Ding & Ramirez (1982) Nucleic Acids Res. 10, 5925-5934] coding for a large portion of helical region including the complete CB7 and CB3 CNBr-cleavage peptides. This cDNA construct was ligated into the transcription vector pSP65, and cell-free translation of the mRNA transcribed from the pSP65 plasmid was performed with a rabbit reticulocyte lysate system. After CNBr cleavage of the hybrid protein translation products, the collagen CB7 and CB3 peptides were resolved by two-dimensional electrophoresis into the same multiple charged forms whether the mRNA was produced from the cDNA construct or was extracted from normal fibroblast cultures. This result demonstrated that the multiple peptide spots were not due to polymorphic mRNA species. The heterogeneity must result from some uncharacterized specific post-translational modification or chemical alterations during sample preparation. This method of expression and analysis of proteins from cDNA clones should be of considerable use in the identification and characterization of clones that code for mutant proteins.