An assay for Clostridium pasteurianum apoferredoxin messenger ribonucleic acid (mRNA) was developed, based on the synthesis of the protein in vitro. Quantitation of apoferredoxin synthesis was accomplished by trypsinization of the cell-free incubation labeled with 3H- or 14C-labeled amino acids, separation of the products by SDS-urea polyacrylamide gel electrophoresis, and excising and counting the NCS-solubilized gel band corresponding to the unique 52-amino acid tryptic peptide derived from apoferredoxin. Its synthesis was shown to be RNA dependent, and was optimized with respect to several parameters of the in vitro protein-synthesizing system. The specificity of the assay was examined with RNA from Clostridium acidi urici, a related species the ferredoxin of which does not yield the 52-amino acid tryptic peptide, and by the use of [3H]leucine, which is not present in C. pasteurianum apoferredoxin. By these methods, the overestimation of apoferredoxin synthesis due to the comigration of fragments from other in vitro products with the legitimate apoferredoxin-derived peptide could be accounted for. The apoferredoxin mRNA was partially purified by the sequential zonal sucrose gradient centrifugation of total RNA followed by Sephadex G-200 chromatography of the enriched RNA, after which a fraction was obtained in which apoferredoxin mRNA was 20-fold enriched. The enriched RNA fraction can now be used for further purification of the apoferredoxin-coding sequences by cloning procedures.