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The purification of motile and virulent Treponema pallidum, Nichols strain, from rabbit testicular tissue is reported. Suspensions of T. pallidum were overlayed onto 20-ml cushions of 43% Percoll and in-situ density gradients were formed by centrifugation at 34,800 g for 30 min. Gradient fractionation indicated that T. pallidum banded at a density of 1.051 g/cc3 and that soluble proteineous testicular components remained in the upper portion of the gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the removal of host testicular and serum components. Purified suspensions of T. pallidum were > 95% actively motile and fully virulent, and >50% motility could be maintained in vitro for up to five days. As determined by electron microscopy, Percollpurified T. pallidum was structurally unaltered and contained much less tissue debris than did crude extracts or T. pallidum prepared by differential centrifugation. The Percoll purification method has been applied successfully to physiology, recombinant DNA, and antigenic structure studies, and to the preparation of antigen for the fluorescent treponemal antibody-absorbed (FTA-Abs) test for syphilis.