Chlamydia psittaci is a significant cause of conjunctivitis in cats, but can be difficult to diagnose owing to the small number of organisms in conjunctival swabs. In the United Kingdom laboratory diagnosis is based on three techniques: isolation of the infectious organism, amplification of chlamydial DNA by the polymerase chain reaction (PCR) or the detection of anti-chlamydial antibodies by immunofluorescence assay. To determine the most sensitive method these techniques were compared in the field. The PCR based on previously published protocols was less sensitive than isolation, but by modifying the protocol its sensitivity was increased by a factor of 25 to 1250 and it was then more sensitive than isolation. The modified PCR detected chlamydia in samples containing non-infectious organisms. Serology was of limited use in predicting which cats shed C psittaci although seronegative cats were negative by PCR and isolation. The modified PCR was the most sensitive and robust method for confirming C psittaci infection in cases of conjunctivitis in pet cats.