Delayed post-operative endophthalmitis is a complication of modern cataract extraction and posterior chamber lens implantation. Propionibacterium acnes has been isolated in a few such cases but the majority are culture-negative, compounding surgical and medical management decisions. A method of detecting bacterial, and specifically P. acnes, DNA by the polymerase chain reaction (PCR) directed at 16S rDNA is reported. Nested PCR with universal eubacterial primers complimentary to regions of 16S rDNA conserved sequences detected 50 fg of bacterial DNA spike in normal vitreous. Nested PCR with P. acnes primers detected 10 fg of DNA. Vitreous samples from 29 patients undergoing vitrectomy for reasons unrelated to infection and 23 samples from 19 patients with delayed post-operative endophthalmitis were analysed. Four (14%) of 29 normal individuals and 17 (74%) of 23 delayed cases gave positive results with universal eubacterial primers. None of 29 and eight of 23 samples gave positive results with P. acnes primers. The 14% positive rate with universal primers in non-infected cases may limit their use in delayed post-operative endophthalmitis. PCR detection of bacterial DNA with specific primers from vitreous samples may prove a useful means of diagnosing delayed post-operative endophthalmitis and facilitating management decisions when conventional bacterial culture is negative.