Members of the genus, Treponema, require long-chain fatty acids for growth in vitro. Serum, added to culture medium, provides a source of long-chain fatty acids. These fatty acids, however, are esterified to triglycerides, phospholipids, and cholesterol. In this study, the major pathways of complex lipid catabolism in five species of Treponema were investigated. Lipase, the enzyme which hydrolyzes triglycerides, was demonstrated using four lipid substrates in five species. Chromatographic data demonstrated that, during growth, treponemes completely utilized lysophosphatidylcholine, present in serum-supplemented culture medium, while phosphatidylcholine and phosphatidylinositol were not utilized. Whole cultures and cell free extracts were examined for the presence of specific enzymes of phospholipid catabolism. Phospholipase B and glycerophosphorylcholine diesterase activities were demonstrated in five species of Treponema. Acid phosphatase activity was demonstrated in two species. Utilization of L-α-glycerophosphate and glycerol, products of phospholipid catabolism, was examined. Glycerol was utilized from culture medium by one species, while α-glycerophosphate was utilized by four species. Phospholipases A, C, and D, α-glycero-phosphate dehydrogenase, and alkaline phosphatase activities were not found in five species of Treponema. Based on the specific enzymes demonstrated in this study, two pathways of phospholipid catabolism are proposed. / M.S.