A Gram-positive, rod-shaped, non-spore-forming bacterium (strain K362ᵀ) was isolated from a deep-water marine sponge collected off the coast of Curacao in the Netherlands Antilles. On the basis of 16S rRNA gene sequence similarities, strain K362ᵀ was shown to belong to the genus Tsukamurella, being most closely related to Tsukamurella pulmonis (99.2 %), Tsukamurella tyrosinosolvens (98.9 %), Tsukamurella strandjordii (98.8 %), Tsukamurella pseudospumae (98.8 %) and Tsukamurella spumae (98.8 %). A combination of the substrate utilization patterns, the fatty acid and mycolic acid profiles and the DNA–DNA hybridization results supported the affiliation of strain K362ᵀ to the genus Tsukamurella and enabled the genotypic and phenotypic differentiation of strain K362ᵀ from the seven recognized Tsukamurella species. Strain K362ᵀ therefore represents a novel species of the genus Tsukamurella, for which the name Tsukamurella spongiae sp. nov. is proposed. The type strain is K362ᵀ (=DSM 44990ᵀ = NRRL B-24467ᵀ). ; Florida Atlantic University. Harbor Branch Oceanographic Institute contribution 1654 ; This manuscript is an author version with the final publication available and may be cited as: Olson, J. B., Harmody, D. K., Bej, A. K., & McCarthy, P. J. (2007). Tsukamurella spongiae sp. nov., a novel actinomycete isolated from a deep-water marine sponge. International Journal of Systematic and Evolutionary Microbiology, 57(7), 1478-1481. doi:10.1099/ijs.0.64837-0