A Gram-positive, rod-shaped, non-spore-forming bacterium (strain K362(T)) was isolated from a deep-water marine sponge collected off the coast of Curaçao in the Netherlands Antilles. On the basis of 16S rRNA gene sequence similarities, strain K362(T) 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 K362(T) to the genus Tsukamurella and enabled the genotypic and phenotypic differentiation of strain K362(T) from the seven recognized Tsukamurella species. Strain K362(T) therefore represents a novel species of the genus Tsukamurella, for which the name Tsukamurella spongiae sp. nov. is proposed. The type strain is K362(T) (=DSM 44990(T)=NRRL B-24467(T)).