We used monoclonal antibodies capable of distinguishing serovars of Chlamydia trachomatis to compare 314 cervical isolates with 150 rectal isolates from homosexual men. The isolates were obtained from patients attending a sexually transmitted diseases clinic over a two-year study period. The serovar distribution of cervical and rectal isolates differed significantly. Serovar D/D' was found in 53% of the rectal isolates but in only 18% of cervical isolates (P less than .0001). Serovar E was the predominant serovar in cervical isolates (32%) but was found in only 6% of rectal isolates (P less than .0001). Serovars B, I/I', H, and K were isolated from 2%-7% of cervical specimens but were not found in rectal isolates. There was a significant decline in the proportion of rectal infections caused by serovar D/D' over the study period, and clustering of infections caused by other serovars was observed. Serotyping using monoclonal antibodies provides a powerful tool for investigating the epidemiology of sexually transmitted C. trachomatis infections.