Treponema pallidum (Nichols virulent strain) was incubated under 75% N2 + 20% H2 + 5% CO2 in prereduced serum-free modified Eagle-Richter medium supplemented with different concentrations of various long-chain fatty acids complexed with fatty acid-free bovine serum albumin. Motility retention was greater in medium with oleic acid containing 15 rather than 2 mg of albumin per ml. Palmitic, stearic, oleic, or linoleic acid alone caused rapid loss of motility at concentrations as low as 5 microgram/ml. Elaidic acid (92 microgram/ml) alone had no effect on motility. Various combinations of saturated plus unsaturated fatty acids did not inhibit motility retention or were less inhibitory than either of the individual fatty acid components. The combination of palmitic plus oleic acids was least toxic. Rapid loss of motility occurred with pairs of unsaturated or saturated fatty acids, or with Tween 40, 60, or 80, alone or combined. Autoxidation of oleic acid resulted in decreased toxicity for T. pallidum but increased toxicity for baby hamster kidney cells.