A double-staining procedure for the fluorescent treponemal antibody-absorption test, using fluorescein isothiocyanate as a label for the class-specific anti-human globulin and tetramethylrhodamine isothiocyanate as a label for a counterstain reagent, has been described. This method requires the addition of a KP560 barrier filter, with a microscope equipped with vertical illumination, to exclude the rhodamine emission in reading the fluorescein fluorescence. The present study evaluated reversing the dye label for each conjugate in the double-staining procedure, thus eliminating the need for the KP560 filter. It also considered the possibility of shortening the counterstaining time and compared various methods for preparing antigen slides in an attempt to establish a method that increases the number of treponemes per microscopic field. The results indicate that a rhodamine-labeled class-specific anti-human globulin as a primary stain, and a fluorescein-labeled anti-treponemal globulin as a counterstain, provide an acceptable method for performing the fluorescent treponemal antibody-absorption double-staining procedure. Nonfixed antigen slides were held for 16 days in a desiccator or stored in plastic bags with silica gel for 3 weeks; then, with methanol fixation, they were used satisfactorily in the double-staining procedure. A shortened incubation time for the counterstain allowed more rapid slide processing.