The sensitivity and specificity of the Bio-EnzaBead test for syphilis and the fluorescent treponemal antibody-absorption (FTA-ABS) test were determined by examining 262 serum samples, including 202 serum samples from patients with confirmed syphilis in various stages. Overall correlation with patient history was 95.8% with both tests. False-negative Bio-EnzaBead tests occurred in 9 of 86 (10.5%) cases of late-latent syphilis (greater than 2 years) and in 1 of 38 (2.6%) cases for which the stage of disease could not be determined. False-negative FTA-ABS tests occurred in 5 of 86 (5.8%) cases of late-latent syphilis (greater than 2 years) and in 2 of 38 (5.3%) cases for which the stage of disease could not be determined. One false-positive test occurred with Bio-EnzaBead, and the cause could not be determined. The reproducibility of the Bio-EnzaBead test was excellent when spectrophotometric readings were calibrated against either air or substrate blanks. The Bio-EnzaBead test for syphilis is a suitable alternative to the FTA-ABS test.