Large numbers of actinomycetes provisionally assigned to the genus Amycolatopsis were isolated from soil samples using a dilution plate procedure and three novel media designed to be selective for members of this genus. A set of genus-specific oligonucleotide primers developed for the rapid identification of unknown Amycolatopsis strains was used to determine whether representative strains taken from the selective isolation plates produced the diagnostic amplification product. The 175 isolates which tested positive were assigned to major, minor and single-membered colour-groups. Representatives of the major colour-groups had a profile of chemical and morphological properties consistent with their assignment to the genus Amycolatopsis; strains taken to represent most of these taxa formed distinct phyletic lines in the Amycolatopsis 16S rRNA gene tree. Many of the representative isolates were closely related to either Amycolatopsis mediterranei or to Amycolatopsis orientalis or formed new evolutionary lines in either the Amycolatopsis orientalis or Amycolatopsis methanolica 16S rRNA subclades. It is evident that the genus Amycolatopsis is grossly underspeciated.