We compared tissue culture with Papanicolaou-stained cervical smears, endocervical cytologic smears stained with immunofluorescent monoclonal antibody, and a direct immunofluorescent stain of cervical specimens (MicroTrak) for detection of Chlamydia trachomatis in cervical specimens. Fifty-one (21%) of 245 women had positive cultures for C. trachomatis, 14 (27%) of whom had clinical evidence of cervicitis. With the criteria of intracytoplasmic coccoid inclusion bodies within metaplastic cells, 45 (34%) of 130 Papanicolaou smears were read as suggestive for C. trachomatis. Seventeen of the 45 positive Papanicolaou smears were positive on culture and 28 were negative (sensitivity 54%, specificity 71%). In contrast, 48 of 51 women with positive cultures and one woman with a negative culture had positive immunofluorescent-stained cytologic smears (sensitivity 94%, specificity 99%, with positive predictive value of 98%). Similarly, 47 of 51 women with positive cultures also had positive results with MicroTrak direct immunofluorescent stain, with only one positive specimen in 196 women with negative cultures (sensitivity 92%, specificity 99%, with positive predictive value of 98%). This study demonstrates that immunofluorescent staining of cervical specimens or of cytologic smears is a more sensitive and specific method than routine Papanicolaou smear for detection of chlamydia infection in a high-prevalence population.