Cell-free protein synthesis offers rapid access to proteins that are selectively labelled with [15N]amino acids and suitable for analysis by NMR spectroscopy without chromatographic purification. A system based on an Escherichia coli cell extract was optimized with regard to protein yield and minimal usage of 15N-labelled amino acid, and examined for the presence of metabolic by-products which could interfere with the NMR analysis. Yields of up to 1.8 mg of human cyclophilin A per mL of reaction medium were obtained by expression of a synthetic gene. Equivalent yields were obtained using transcription directed by either T7 or tandem phage lambdapR and pL promoters, when the reactions were supplemented with purified phage T7 or E. coli RNA polymerase. Nineteen samples, each selectively labelled with a different 15N-enriched amino acid, were produced and analysed directly by NMR spectroscopy after ultracentrifugation. Cross-peaks from metabolic by-products were evident in the 15N-HSQC spectra of 13 of the samples. All metabolites were found to be small molecules that could be separated readily from the labelled proteins by dialysis. No significant transamination activity was observed except for [15N]Asp, where an enzyme in the cell extract efficiently converted Asp-->Asn. This activity was suppressed by replacing the normally high levels of potassium glutamate in the reaction mixture with ammonium or potassium acetate. In addition, the activity of peptide deformylase appeared to be generally reduced in the cell-free expression system.