We studied 170 strains of Actinomadura, Nocardiopsis, and related taxa. The overall similarities of the test strains were determined by comparing 120 unit characters, using the simple matching and pattern coefficients. Clustering was achieved by using the unweighted pair group method with averages. Cluster composition was not markedly affected by the coefficient used or by test error (estimated at 4.9%). The numerical data, together with results from previous chemical and genetic studies, showed that the genus Actinomadura is markedly heterogeneous, Strains received as Actinomadura fell into two large aggregate groups. One, Actinomadura sensu stricto, contained Actinomadura citrea, Actinomadura coerulea, Actinomadura coeruleoviolacea, Actinomadura cremea, Actinomadura livida, Actinomadura madurae, Actinomadura malachitica, Actinomadura pelletieri, and Actinomadura verrucosospora. Actinomadura kijaniata was not studied, but other genetic data suggest that it should also be included in this group. The other aggregate group encompassed Actinomadura ferruginea, Actinomadura pusilla, Actinomadura roseola, Actinomadura roseoviolacea, Actinomadura rubra, and Actinomadura salmonea. The representatives of Actinomadura helvata, Actinomadura libanotica, Actinomadura luteofluorescens, Actinomadura spadix, and Actinomadura spiralis studied formed distinct clusters separate from both of the aggregate groups. Nocardiopsis strains formed a cluster clearly distinguished from the clusters containing the Actinomadura strains.The genus Actinomadura (36) was introduced for sporoactinomycetes that form branched, usually stable substrate mycelia with walls containing meso-2,6-diaminopimelic acid but lacking arabinose and galactose (36, 38). This taxon was cited as a genus incertae sedis in Bergey's Manual of Determinative Bacteriology, 8th ed. (40), but was included on the Approved Lists of Bacterial Names (52). The genus originally contained three species Actinomadura dassonvillei, Actinomadura madurae, and Actinomadura pelletieri, and was classified in the family Thermoactinomycetaceae (36). It was subsequently transferred to the family Thermomonosporaceae because endospores were not produced (ll), but it is now thought to be related to genera with chemotype I11 walls, notably Microbispora and Microtetraspora (3,20,24,64). The size of the genus Actinomadura has increased so that 34 species are currently recognized or have been proposed (31, 32,42,46,52,60), primarily on the basis of morphology and wall chemotype. However, there is some evidence that both A. madurae and A . pelletieri are heterogeneous (4,7, 19). The third original species, A. dassonvillei, formed the basis of the genus Nocardiopsis (41), the integrity of which is supported by an abundance of chemical, genetic, morphological, and numerical phenetic data (5, 12, 24, 35,64).The genus Actinomadura encompasses a range of morphological types (35, 65) and has also been shown to be heterogeneous on the basis of fatty acid (2, 12), menaquinone (5, 12, 67), polar lipid (37, ...