Specific immunofluorescence staining was applied to the study of the localization, distribution, and growth of Thermoplasma acidophilum in its natural habitat, the coal refuse pile. Different antigenic groups of T. acidophilum could be isolated from the same refuse pile, and the same antigenic groups were isolated from piles from different geographical areas. No correlation could be established between the antigenic groups and the pH or temperature of the habitats. Brightly fluorescing cells of T. acidophilum were detected on microscope slides buried in contact with the coal refuse material or immersed in the water in the stream draining a refuse pile. T. acidophilum grew when inoculated into either coal refuse material and/or an aqueous extract of coal refuse when incubated at its optimal temperature of 55 C, but not when incubated at room temperature or 37 C. The coal refuse pile appears to be a primary habitat for T. acidophilum.