The taxonomic position of two soil isolates, strains A288(T) and A290(T) [provisionally assigned to the genus Actinomadura] was clarified in a polyphasic study. The organisms showed a combination of chemotaxonomic and morphological properties typical of actinomadurae. They also formed distinct phyletic lines in the 16S rRNA Actinomadura gene tree; strain A288(T) was associated with A. nitritigenes whereas strain A290(T) was closely related to a group that consisted of A. citrea, A. coerulea, A. glauciflava, A. luteofluorescens and A. verrucosospora. Strains A288(T) and A290(T) showed key phenotypic features which readily distinguish them from one another and from representatives of related validly described species of Actinomadura. It is proposed that the two organisms be classified as new species of the genus Actinomadura. The names proposed for the new taxa are Actinomadura mexicana (A290(T) = DSM 44485(T) = NRRL B-24203(T)), and Actinomadura meyerii (A288(T) = DSM 44485(T) = NRRL B-24203(T)).