Expression of proteins from cDNA clones by transcription and translation in vitro can be an attractive option for analytical applications that do not require significant quantities of product. Our laboratory has been interested in this technique because the translation of nucleic acid into protein sequence allows use of the unsurpassed resolving power of 2D protein electrophoresis to analyze complex mixtures of gene products. Thus, typically we might compare two cell types that differ in some phenotypic trait, initially by translation of mRNA purified from both sources and 2D electrophoresis of the translation products. If a 2D gel spot varies in an interesting manner between the cell types, we convert the population of mRNA molecules expressed in the positive cell type into a population of cDNA clones, then search among the clones for one that produces the spot of interest after in vitro transcription and translation. The 2D gel spot is used only as the diagnostic signature of an mRNA species, and does not need to be physically handled itself to permit the recovery of cognate cDNA clones; in this way, this approach differs radically from strategies based on microscale sequencing. However, other investigators may wish to use cell-free expression to monitor mutagenesis, epitope mapping or the biological activity of already isolated clones encoding known proteins. The methods described here can be applied equally well to single clones and to complex mixtures of clones.