A novel actinomycete strain, designated GKU 128T, isolated from the roots of an Indian oak tree [Barringtonia acutangula (L.) Gaertn.] at Khao Khitchakut district, Chantaburi province, Thailand, was characterized by using a polyphasic approach. The strain formed a branched substrate and aerial mycelia which differentiated into straight to flexuous chains of smooth-ornamented spores. Analysis of the cell wall revealed the presence of meso-diaminopimelic acid and N-acetylmuramic acid in the peptidoglycan. The whole-cell sugars were glucose, madurose, mannose, rhamnose and ribose. Mycolic acids were absent. The major phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositolmannoside. The predominant menaquinones were MK-9(H6), MK-9(H8), MK-9(H0) and MK-9(H4). The major fatty acids were C16 : 0, C18 : 1ω9c and 10-methyl C18 : 0 (tuberculostearic acid). The genomic DNA G+C content was 70.5 mol%. Based on 16S rRNA gene sequence analysis, strain GKU 128T was closely related to the type strains of Actinomadura nitritigenes NBRC 15918T (99.2 % sequence similarity) and Actinomadura fibrosa JCM 9371T (98.7 %). The levels of DNA-DNA relatedness between strain GKU 128T and the closely related type species were less than 19 %. On the basis of phenotypic and genotypic characteristics, strain GKU 128T could be distinguished from its closely related type strains and represents a novel species of the genus Actinomadura, for which the name Actinomadura barringtoniae sp. nov. (=TBRC 7225T=NBRC 113074T) is proposed.