Two actinomycete strains, 32U-2T and 32U-4, were isolated from a prairie soil in Minnesota and subjected to characterization by means of polyphasic taxonomy. The 16S rRNA gene sequences were determined following PCR amplification and cloning. A phylogenetic analysis, based on comparative analysis of 16S rRNA gene sequences, indicated that the organisms consistently formed a well-separated, distinct sub-branch within the radiation of the genus Amycolatopsis of the family Pseudonocardiaceae. The levels of 16S rRNA sequence similarity between the isolates and the type strains of recognized Amycolatopsis species ranged from 94.1 to 97.9 %. The highest levels of sequence similarity were found between the isolates and Amycolatopsis coloradensis (97.6-97.9 %), Amycolatopsis alba and Amycolatopsis orientalis (97.3-97.6 %) and Amycolatopsis lurida (97.2-97.5 %). Chemotaxonomic characteristics supported the phylogenetic relationships between the organisms and members of the genus Amycolatopsis. However, a broad range of phenotypic and genetic data revealed that the isolates should be classified as novel species of the genus Amycolatopsis, for which the name Amycolatopsis minnesotensis sp. nov. is proposed. The type strain is 32U-2T (=KCCM 42246T = NRRL B-24435T).