Definitive diagnosis of early syphilis currently requires dark-field microscopy and/or a newly reactive serologic test for syphilis. The efficacy of dark-field microscopy depends on the availability of a microscope, the skill of the clinician in obtaining a specimen, and the expertise of the microscopist. Serologic diagnosis may be affected by the delay between the appearance of the primary chancre and the onset of serologic reactivity. We used a pathogen-specific fluorescein-conjugated monoclonal antibody to examine lesion exudates from 128 consecutive patients and compared these data with results of dark-field microscopy, the rapid plasma reagin (RPR) test, and the fluorescent treponemal antibody-absorbed (FTA-Abs) test. The monoclonal antibody test demonstrated Treponema pallidum in 48 (73%) of 66 patients with infectious syphilis, while dark-field microscopy was positive for 52 (79%) of 66 patients. None of 62 patients without syphilis was positive by either test. The FTA-Abs test was reactive for 61 patients (92%) of the 66 with infectious syphilis. Thus the fluorescent monoclonal antibody test for detection of T. pallidum in direct smears is as sensitive and specific as dark-field microscopy for the diagnosis of infectious syphilis. It has the potential to provide a convenient, accurate means for definitive diagnosis of genital ulcer disease by health care personnel without ready access to dark-field microscopy. © 1987 American Sexually Transmitted Diseases Association.