A molecular biological method of detecting and serotyping Chlamydia trachomatis (C. trachomatis) directly from conjunctival specimens by polymerase chain reaction (PCR) was developed and applied to the diagnosis of inclusion conjunctivitis. We amplified 1.2 kbp DNA fragments of ompA gene from 15 reference strains of C. trachomatis by two-step PCR using two pairs of primers. Restriction fragment length polymorphism analysis using a combination of three endonucleases (HinfI, HindIII and HhaI) completely differentiated 13 of the 15 serovars; the exceptions were B and Ba. We then used this method for 18 strains of C. trachomatis isolated from Japanese patients with inclusion conjunctivitis, serotyping them into six groups: D (5/18), G (5/18), E (3/18), H (2/18), F (1/18), and K (1/18). In our comparison of cell culture isolation with PCR analysis of 38 conjunctival swabs from 35 patients in Sapporo with follicular conjunctivitis, 8 were positive in culture isolation and were also positive in two-step PCR. Twenty-five of 26 strains (the 18 isolated strains and 8 strains amplified by two-step PCR) were genotyped to D, G, H, E, F, and K. One isolated strain could not be identified. The C. trachomatis which causes inclusion conjunctivitis in Japan appears to have a distribution of serovars similar to that of the sexually transmitted diseases.