An enzyme immunoassay (Chlamydiazyme) for detecting Chlamydia trachomatis was evaluated on genital specimens from 96 men and 272 women attending a clinic for sexually transmitted diseases (STD clinic). Compared with a direct immunofluorescence test for chlamydial elementary bodies, the enzyme immunoassay had a sensitivity of 58% on specimens from men, a specificity of 90%, a positive predictive value of 93%, and a negative predictive value of 88%; the assay had a sensitivity of 67% on specimens from women, a specificity of 89%, a positive predictive value of 63% and a negative predictive value of 90%. Immunofluorescence provided the most stringent test for the performance of the enzyme immunoassay as values were improved a little when a cell culture procedure was used for comparison. Further evidence for the lack of sensitivity was the detection of elementary bodies, sometimes in large numbers, in the enzyme immunoassay buffer of 13 of 19 specimens that had given a negative enzyme immunoassay result and the finding in comparative titrations of four laboratory strains that the enzyme immunoassay was at least 100-fold less able to detect chlamydiae than either immunofluorescence or the cell culture procedure. Lack of specificity may be associated with the finding that the enzyme immunoassay antibody reacted with strains of Acinetobacter calcoaceticus, Escherichia coli, Gardnerella vaginalis, Neisseria gonorrhoeae and group B streptococci. The enzyme immunoassay was not considered to be sufficiently sensitive, specific, or reproducible for routine use.