Several laboratories have demonstrated that the polymerase chain reaction (PCR) is more sensitive than culture or enzyme immunoassay (EIA) for detecting Chlamydia trachomatis in genitourinary tract specimens when various DNA targets are used for amplification, including the cryptic plasmid, major outer membrane protein (MOMP), or rRNA genes. We compared the performances of five different PCR assays, including assays with two plasmid, two MOMP, and one rRNA targets, by amplifying serial dilutions of C. trachomatis DNA and testing genitourinary tract specimens. By using published procedures, two different plasmid primers had sensitivities of 0.1 fg for C. trachomatis plasmid DNA and 10 fg for total cellular DNA. The sensitivities of the assays with the two MOMP primers were 0.1 and 10 pg, and the sensitivity for the assay with the rRNA primers was 1 pg for cellular DNA. Both plasmid-based assays detected 38 of 38 confirmed Chlamydiazyme-positive specimens, whereas the assays with the MOMP and rRNA primers detected 36 of 38 and 29 of 38 confirmed Chlamydiazyme-positive specimens, respectively. Six of 18 Chlamydiazyme-negative specimens collected from individuals whose specimens were positive by culture or immunofluorescence were positive by both plasmid-based PCRs; 4 of these were positive by PCR with the MOMP primers and 3 were positive by PCR with the rRNA primers. The results obtained with both purified DNA and genitourinary tract specimens indicated that the plasmid-based PCRs are more sensitive than bacterial chromosome-based PCRs for detecting C. trachomatis.