Murine anti-Treponema pallidum monoclonal antibodies were employed in studies on sensitivity and specificity of binding to examine their potential for use in the detection of low numbers of pathogenic treponemes present in various body fluids. Monoclonal antibodies were used as a primary antibody source in a solid-phase immunoblot assay system. All monoclonal antibodies assayed were capable of detecting ca. 1.0 X 10(3) to 2.5 X 10(3) treponemes. Of 13 monoclonal antibodies examined, 3 were able to detect 10(3) virulent treponemes, and 1 of these antibodies was able to reveal the presence of as few as 500 organisms. Western blot analyses showed that all anti-T. pallidum monoclonal antibodies exhibiting high sensitivities for the detection of T. pallidum cells were directed against an abundant, 47,000-dalton surface-exposed antigen of the organism (S. A. Jones, K. S. Marchitto, J. N. Miller, and M. V. Norgard, Abstr. Annu. Meet. Am. Soc. Microbiol. 1984, B173, p. 46; K. S. Marchitto, S. A. Jones, and M. V. Norgard, Abstr. Annu. Meet. Am. Soc. Microbiol. 1984, B182, p. 48). Differences in binding properties of the various monoclonal antibodies were most likely a reflection of differential binding affinities or their specificities for different epitopes on the 47,000-dalton surface antigen. With two possible exceptions, the monoclonal antibodies tested reacted specifically with T. pallidum, either purified or found within a high-contaminating tissue background, and not with Treponema phagedenis biotype Reiter, Haemophilus ducreyi, Neisseria gonorrhoeae, herpes simplex virus type 2, or normal rabbit testicular tissue. The high sensitivity and specificity exhibited by these anti-T. pallidum monoclonal antibodies make them excellent candidates for employment in new syphilis or other treponemal diagnostic tests designed to detect very low numbers of pathogenic treponemes in lesion exudates or other body fluids.