The extraction of Treponema pallidum antigen with sodium desoxycholate, based on a previously described procedure (J. Portnoy and H.J. Magnuson, J. Immuno. 75:348-355, 1955), was used in an enzyme-linked immunosorbent assay (ELISA) test for syphilis. The antigen was prepared from T. pallidum street strain no. 14, and its overall sensitivity and specificity was compared with those of sonicated antigen preparations made with phosphate-buffered saline. The optimum serum dilution for testing and the significant absorbance reading at 490 nm were selected by examination of quantitative dilutions of 91 sera from presumably normal individuals and 92 sera from syphilitics. The time and temperature of serum and conjugate incubations were also examined. With an absorbance reading of greater than or equal to 0.2 at the 1:80 serum dilution, 88 (95.8%) of 92 sera from syphilitics were reactive in the ELISA test with desoxycholate-extracted antigen, and 82 (89.1%) were reactive with the sonicated antigen. Only one nonsyphilitic serum was reactive with each antigen. Greater sensitivity without loss in specificity was obtained with longer serum and conjugate incubations. We concluded that an ELISA test with sodium desoxycholate-extracted antigen is more sensitive than and equally specific to an ELISA with sonicated treponemal antigen.