A method that avoids culture was devised to determine serovars of Chlamydia trachomatis. Polymerase chain reaction was used first to amplify a part of the chlamydial genome that included the leader sequence and all four variable domains of the major outer membrane protein (MOMP) of the 15 serovars of C. trachomatis. The amplified DNA was then digested simultaneously with restriction endonucleases AluI and MspI and the resulting fragments separated on 10% polyacrylamide gels. After silver staining, a total of 13 characteristic patterns were observed for the 15 serovars, leaving two ambiguities that were resolved using alternate enzymes. Analysis of 40 clinical isolates revealed patterns indistinguishable from those of the prototype serovars including, unexpectedly, 5 Ba serovars. The same PCR procedure also allowed amplification of the MOMP gene of two avian Chlamydia psittaci and one Chlamydia pneumoniae isolates.