Adenoviruses are associated with endemic and epidemic acute conjunctivitis, large nosocomial outbreaks reflecting virus transmission on unwashed hands or inadequately sterilised ophthalmic instruments. The polymerase chain reaction (PCR) proved more sensitive than antigen detection by immune dot-blot test for the rapid diagnosis of ocular adenovirus infection (sensitivities in a retrospective study 112/123 (91%) versus 72/123 (59%), P < 0.001). Indeed, in a prospective comparison, DNA amplification and virus isolation generated similar numbers of positive results (34 versus 32), though five PCR positive results were possibly false positives. The sensitivity of the PCR was largely independent of adenovirus subgenus or serotype, though reduced sensitivity with subgenus B strains could not be excluded. Specimen preparation for DNA amplification using a simple lysis buffer proved more effective than phenol-chloroform extraction. The immune dot-blot test gave unavoidable false positive results, but with the PCR this problem could be minimized by technical modifications. The PCR could replace antigen detection and virus isolation as the initial test for adenoviruses in conjunctival swabs, with cell culture only being retained for adenovirus serotyping in PCR positive specimens and for other viruses such as herpes simplex.