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.