Purpose: To investigate and compare the use of molecular biology with the use of traditional Gram stain and organism culture for the laboratory diagnosis of postoperative endophthalmitis., Setting: Twenty-four ophthalmology units together with 9 microbiology laboratories and 2 European reference molecular biology laboratories., Methods: A prospective randomized partially masked multicenter cataract surgery study recruited 16 603 patients. This resulted in 29 cases of presumed postoperative endophthalmitis. Gram stain and culture were performed in the local laboratory according to agreed protocols. Samples of aqueous and/or vitreous were transported to the first referenced molecular biology laboratory (Regensburg, Germany) for polymerase chain reaction (PCR) testing, and an extracted aliquot of DNA was then referred to the second laboratory (Alicante, Spain) for PCR., Results: Of the 29 who presented with presumed postoperative endophthalmitis, 20 were classified as proven infective endophthalmitis with positive Gram stain, culture, or PCR. Fourteen patients were culture-positive; all but 1 of these was also positive by PCR. Six patients were positive by PCR but negative by Gram stain or culture. Nine patients were negative by both microbiology and PCR testing., Conclusions: Use of molecular biology technique increased the laboratory rate of identifying the pathogen by 20%, confirming the technique is very useful for the endophthalmitis specimen. Samples of both aqueous and vitreous should be collected and stored at -20 degrees C for PCR at the time of the diagnostic taps.