Both mycelial fragments and protoplasts were successfully employed for mutagenesis of Micromonospora rosaria NRRL 3718, and the results were compared. The optimal conditions and effective procedures for mutagenesis of M. rosaria by a chemical mutagen, N-methyl-N'-nitro-N-nitrosoguanidine, have been determined. Mutation was efficiently induced when mycelial fragments were treated with N-methyl-N'-nitro-N-nitrosoguanidine at a concentration of 0.3 to 0.5 mg/ml in the reaction buffer of pH 7.0. Optimal treatment time was 20 to 40 min. Ampicillin treatment was very effective for enrichment of auxotrophs. Protoplasts showed much higher sensitivity to the lethal effect of N-methyl-N'-nitro-N-nitrosoguanidine. Although protoplasts have some advantage of single cell characteristics, the frequency of auxotrophs obtained was somewhat lower. Up to 4% of the colonies were shown to be auxotrophs under the well-defined conditions. This mutagenesis method with protoplasts or fragmented mycelia (or both) should be applicable to other actinomycetes that have limited or no sporulation.