A large number of mycolate actinomycetes have been recovered from deep-sea sediments in the NW Pacific Ocean using selective isolation methods. The isolates were putatively assigned to the genus Rhodococcus on the basis of colony characteristics and mycolic acid profiles. The diversity among these isolates and their relationship to type strains of Rhodococcus and other mycolate taxa were assessed by Curie point pyrolysis mass spectrometry (PyMS). Three major (A, C, D) and two minor (B, E) groups were defined by PyMS. Cluster A was a large group of isolates recovered from sediment in the Izu Bonin Trench (2679 m); Cluster C comprised isolates from both the Izu Bonin Trench (6390 and 6499 m) and from the Japan Trench (4418, 6048 and 6455 m). These Cluster C isolates showed close similarity to Dietzia maris and this was subsequently confirmed using molecular methods. Cluster D contained isolates recovered from a sediment taken from a depth of 1168 m in Sagami Bay and were identified as members of the terrestrial species Rhodococcus luteus. Clusters B and E had close affinities with members of the genera Gordonia and Mycobacterium. The presence of Thermoactinomyces in certain of the deep-sea sediments studied was indicative of the movement of terrestrial material into the ocean depths. 16S ribosomal RNA gene sequence analyses produced excellent definition of most genera of the mycolata, and indicated that the among the deep sea isolates (1) were novel species of Corynebacterium, Gordonia and Mycobacterium, and (2) a Sea of Japan isolate the phylogenetic depth of which suggests the possibility of a new genus. Polyphasic taxonomic analysis revealed considerable diversity among the deep sea rhodococci and evidence for recently diverged species or DNA groups.