A morpholinepropanesulfonic acid (MOPS)-buffered rich defined medium (RDM) was optimized to support a reproducible 2.6-h doubling time at 35 degrees C for Deinococcus radiodurans R1 and used to gain insight into vitamin and carbon metabolism. D. radiodurans was shown to require biotin and niacin for growth in this medium. A glutamine-serine simple defined medium (SDM) was developed that supported a 4-h doubling time, and this medium was used to probe sulfur and methionine metabolism. Vitamin B-12 was shown to alleviate methionine auxotrophy, and under these conditions, sulfate was used as the sole sulfur source. Phenotypic characterization of a methionine synthase deletion mutant demonstrated that the B-12 alleviation of methionine auxotrophy was due to the necessity of the B-12-dependent methionine synthase in methionine biosynthesis. Growth on ammonium as the sole nitrogen source in the presence of vitamin B-12 was demonstrated, but it was not possible to achieve reproducibly good growth in the absence of at least one amino acid as a nitrogen source. Growth on sulfate, cysteine, and methionine as sulfur sources demonstrated the function of a complete sulfur recycling pathway in this strain. These studies have demonstrated that rapid growth of D. radiodurans R1 can be achieved in a MOPS-based medium solely containing a carbon source, salts, four vitamins, and two amino acids.