The attine ant-microbe system is a quadripartite symbiosis, involving a complex set of mutualistic and parasitic associations. The symbiosis includes the fungus-growing ants (tribe Attini), the basidiomycetous fungi the ants cultivate for food, specialized microfungal parasites (in the genus Escovopsis) of the cultivar, and ant-associated mu tualistic filamentous bacteria that secrete antibiotics specifically targeted to suppress the growth of Escovopsis. In this study, we conduct the first phylogenetic analysis of the filamentous mutualistic bacteria (actinomycetes) associated with fungus-growing ants. The filamentous bacteria present on 3 genera of fungus-growing ants (Acromyrmex, Trachy myrmex, and Apterostigma) were isolated from 126 colonies. The isolated actinomycetes were grouped into 3 distinct morphological types. Each morphological type was specific to the ant genus from which it was isolated, suggesting some degree of host specificity. The phylogenetic position of the 3 morphotypes was estimated using 16S rDNA for representative strains. The 8 isolates of actinomycetes sequenced are in the family Pseudonocardiaceae (Actino mycetales) and belong to the genus Pseudonocardia. Transmission electron microscopy examination of the actino mycete associated with the cuticle of Acromyrmex sp. revealed bacterial cells with an outer electron-dense membrane, consistent with actinomycetes in the genus Pseudonocardia. Ant-associated Pseudonocardia isolates did not form a monophyletic group, suggesting multiple acquisitions of actinomycetes by fungus-growing ants over their evolutionary history.