To assess the utility of PCR-enzyme immunoassay (EIA) for diagnosis of acute infection with Chlamydia pneumoniae, we compared tissue culture, PCR-EIA, direct fluorescent-antibody (DFA) stain, and serology in studies with 56 patients with respiratory symptoms and 80 asymptomatic persons. Thirty-five patients were positive by either culture or PCR-EIA, and 101 were negative by both assays. Thirty specimens from symptomatic patients and one from an asymptomatic patient were culture positive; 23 of these were also PCR-EIA positive. Of the eight culture-positive, PCR-EIA-negative specimens, five were DFA negative and three were DFA positive. Four additional specimens were culture negative and PCR-EIA positive; of these, three were DFA positive and one was DFA negative. When we used culture- and/or DFA-positive results as a reference or "gold standard," the sensitivity and specificity of PCR were 76.5 and 99.0%, respectively. When we used PCR- and/or DFA-positive results as the reference, the sensitivity of culture was 87.5%. On the basis of single acute serum specimens, only 8 of these 35 patients had diagnostic antibody titers. Of the asymptomatic patients, 75% had immunoglobulin G or immunoglobulin M antibody to C. pneumoniae; 15 (18.8%) of these had antibody levels considered to be diagnostic of acute infection. This multicenter study indicates that culture and/or PCR-EIA is more reliable for prompt diagnosis of C. pneumoniae infection than single-point serology alone.