The fluorescent treponemal antibody absorption (FTA-ABS) double-staining (DS) test has been developed for microscopes equipped with incident illumination, and the procedure offers many advantages over the FTA-ABS test when tests are performed with this equipment. In this study, 346 fresh sera, including 35 from patients with syphilis, were evaluated by the FTA-ABS DS test. Parameters for investigation included two readers, each using a different microscope; a new FTA-ABS DS test reporting system; sera heated at 56 degrees C for 30 min versus unheated sera; and sera retested after at least 2 weeks of freezer storage. Agreement for FTA-ABS DS test readings between the two microscopes was 99%. Between-test agreement for the FTA-ABS test with the conventional reporting system and the FTA-ABS DS test with the new reporting system was 95%. Sensitivity calculations based on reactivity for the 35 syphilis sera were 94% for the FTA-ABS DS test and 91% for the FTA-ABS test. Specificity calculations based on non-reactivity of nonsyphilis sera were 98% for the FTA-ABS DS test and 93% for the FTA-ABS test. Differences in percentages appeared to be related to borderline readings in the FTA-ABS test. For example, if the same reporting system was used for the reference FTA-ABS test, the specificity was 97%. When sera were examined within 48 h, no difference was observed in results obtained with heated and unheated sera. Sera frozen for 2 weeks showed comparable results in the FTA-ABS DS test and the FTA-ABS test. These findings strongly support the recommendation that the FTA-ABS DS test be accepted as a confirmatory test for syphilis. The new reporting system for the FTA-ABS DS test would be advantageous for the reference FTA-ABS procedure.