OBJECTIVE: To estimate the prevalence of Chlamydia trachomatis, C. psittaci and C. pneumoniae antibodies in sera from altogether 931 blood donors, patients with symptoms of urethritis, assumed salpingitis and sexually acquired reactive arthritis (SARA), and women with fertility problems. METHODS: IgG antibodies to C. trachomatis, C. psittaci and C. pneumoniae were determined by microimmunofluorescence (MIF) tests. All patients were also tested for genital C. trachomatis infection using direct immunofluorescence (DIF) tests. RESULTS: The DIF-positive cases had a significantly (p < 0.0001) higher prevalence of C. trachomatis antibodies than the DIF negatives, i.e. 88.5% versus 14% in men with urethritis, 94.3% versus 36.4% in women with salpingitis, 66.7% versus 16.7% in SARA patients and 90.6% versus 20.8% in women with fertility problems. Antibody reactivity to all three chlamydial species was found significantly (p < 0.0001) more often in the patient groups and in those with a DIF-confirmed genital C. trachomatis infection than in blood donors. CONCLUSIONS: Presence of serum antibodies to C. trachomatis is tightly associated with the presence of chlamydiae in the genital tract, which also influences the cross-reactivities occurring in the MIF tests between chlamydial species.