Production of coenzyme F420 and its biosynthetic precursor FO was examined with a variety of aerobic actinomycetes to identify an improved source for these materials. Based on fermentation costs, safety, and ease of growth, Mycobacterium smegmatis was the best source for F420-5,6. M. smegmatis produced 1 to 3 micromol of intracellular F420 per liter of culture, which was more than the 0.85 to 1.0 micromol of F420-2 per liter usually obtained with Methanobacterium thermoautotrophicum and approximately 10-fold higher than what was previously reported for the best aerobic actinomycetes. An improved chromatography system using rapidly flowing quaternary aminoethyl ion-exchange material and Florisil was used to more quickly and easily purify F420 than with previous methods.