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Mycoplasma testudineum sp. nov., first cultured from the upper respiratory tract of a clinically ill tortoise (Gopherus agassizii) in the Mohave Desert, was distinguished from previously described mollicutes serologically and by 16S rRNA gene sequence comparisons. It lacks a cell wall; ferments glucose, mannose, lactose and sucrose; does not produce ’film and spots’; does not hydrolyse arginine, aesculin or urea; is sensitive to digitonin; and lacks phosphatase activity. The organism causes chronic rhinitis and conjunctivitis of tortoises. The type strain of M. testudineum is BH29(T) ( = ATCC 700618(T) = MCCM 03231(T)).