We describe a procedure which permits essentially full recovery of physiologically active Treponema pallidum from rabbit testicular extracts and greatly reduces contaminating tissue material. Such preparations were employed for investigations of the ability of T. pallidum to catabolize glucose and fatty acids. Radiorespirometric studies revealed that glucose and pyruvate, but not oleate or palmitate, could be degraded to CO2. The use of differentially labeled glucose, in conjunction with enzymatic analyses, indicated that glucose was catabolized by a combination of the Embden-Meyerhoff-Parnas and hexose monophosphate pathways. Pyruvate was degraded to CO2 from only the carboxyl position, suggesting the absence of a functioning Krebs cycle; this was substantiated by additional enzyme analyses and radiorespirometric experiments. Oleate and palmitate were incorporated but not catabolized by beta-oxidation. Glucose, although catabolized, was not incorporated. The potential significance of these findings is discussed.