The accuracy of the routine Papanicolaou (Pap) smear in diagnosis of Chlamydia trachomatis infection of the cervix has stimulated controversial reports over the years. In the present study Pap smears from 100 women, evenly divided between culture-positive and culture-negative for C. trachomatis, who had been seen in a single university clinic and cultured in the same laboratory were reviewed by two cytologists. With use of typical inclusions as evidence of chlamydial infection, the sensitivity of the Pap smear was 8.3%. Review of another series of 40 Pap smears that had been reported previously as positive for C. trachomatis showed only 25 with inclusions, an observation suggesting that routine Pap smears are being overread for chlamydial infection. Attempts to improve the sensitivity by immunoperoxidase staining were unsuccessful. Therefore, if strict cytologic criteria are used, C. trachomatis infections may be diagnosed on Pap smears, but the sensitivity will be low.