The menaquinones of 141 actinomycetes representing the genera Caseobacter, Mycobacterium, Nocardia, Rhodococcus and some related taxa lacking mycolic acids were examined by mass spectrometry. The mycolic acid-containing strains were assigned to four groups on the basis of the predominant isoprenologue detected: Rhodococcus coprophilus, R. equi, R. erythropolis, R. globerulus, R. rhodnii, R. rhodochrous and R. ruber contained dihydrogenated menaquinones with eight isoprene units; Nocardia asteroides, N. brasiliensis, N. carnea, N. otitidis-caviarum and N. transvalensis contained tetrahydrogenated menaquinones with eight isoprene units; Caseobacter polymorphus, R. bronchialis, R. rubropertinctus and R. terrae and representatives of twenty-one approved species of Mycobacterium contained dihydrogenated menaquinones with nine isoprene units; a single strain of 'Mycobacterium album', contained unsaturated menaquinones with nine isoprene units. Actinomycetes containing meso-diaminopimelic acid, arabinose and galactose in the wall peptidoglycan but lacking mycolic acids were recovered in two groups: tetrahydrogenated menaquinones with eight isoprene units were the main components from 'Nocardia' autotrophica and Pseudonocardia thermophila whereas Saccharopolyspora hirsuta and Pseudonocardia spp. contained tetrahydrogenated menaquinones with nine isoprene units. Promicromonospora citrea and 'skin coryneforms' with LL-diaminopimelic acid and glycine in the wall peptidoglycan also contained tetrahydrogenated menaquinones with nine isoprene units as the major isoprenologue. In contrast, representatives of the genera Kitasatoa, Microellobosporia, Streptomyces and Streptoverticillium were characterized by the presence of complex mixtures of tetra-, hexa- and octa-hydrogenated menaquinones with nine isoprene units. The menaquinone data correlate well with other developments in actinomycete systematics and confirm earlier suggestions that menaquinone analyses are of value in both the classification and identification of actinomycetes. Indeed, the data suggest that minimal descriptions of wall chemotype IV taxa should ideally include information on menaquinone composition.