The maintenance of Treponema pallidum motility was investigated in an acellular medium based on T. pallidum immobilization test medium. The acellular medium contained cysteine, glutathione, thioglycollate and dithiothreitol as reducing agents and had a redox potential of -275 +/- 25 mV at pH 7.3. In an atomosphere containing 3% O2, motile treponemes survived four times longer when calf serum and bovine serum albumin were added to the medium. The selective omission of glutathione and, particularly, thioglycollate prolonged the survival of motile treponemes almost fivefold. In addition, stored medium, in which thioglycollate had become inactive, sustained motile treponemes for longer than did freshly prepared medium. Thus, thioglycollate is toxic for the organisms. It may be omitted from the medium because low redox potentials can be achieved without it.