A new enzyme-linked immunosorbent assay with Treponema pallidum antigen bound to ferrous metal beads (Syphilis Bio-EnzaBead; Litton Bionetics Laboratory Products) was compared with the standard fluorescent treponemal antibody-absorption test for syphilis. Bio-EnzaBead and fluorescent treponemal antibody-absorption tests were done on 218 specimens from documented cases of syphilis, on 315 sera from individuals with diseases other than syphilis, and on sera submitted to a public health laboratory for premarital (304 specimens) or diagnostic (501 specimens) tests for syphilis. Agreement between the Bio-EnzaBead and reference tests ranged from 93.0% for sera for the diagnostic test to 99.5% for sera from patients with syphilis. The overall agreement among the 1,338 sera tested was 96.3%. The reproducibility of the Bio-EnzaBead test with 60 coded sera of graded reactivity was 97%. The test is easy to perform, the indicator results are clear and unequivocal, and the findings are comparable to those of the fluorescent treponemal antibody-absorption test.