A study was performed to evaluate nested PCR (nPCR) versus viral cultures as method and tear film versus corneal scrapings as specimen in the diagnosis of viral keratoconjunctivitis. Tear film specimens were taken from both eyes and corneal scrapings from the affected eye only in 17 patients with suspected viral keratoconjunctivitis. In 15 of the 17 patients the viral agent of the infection could be detected: 11 patients had herpes simplex virus type 1, two varicella-zoster virus, one both herpes simplex virus type 1 and varicella-zoster virus, and one adenovirus. Overall there was no significant difference between the detection rate for corneal scrapings (85%) and tear film (75%). In both types of specimens nPCR showed a higher detection rate than viral cultures (corneal scrapings: 87.5% vs 31.25%; tear film: 75% vs 12.5%; P 0.05). For the diagnosis of keratoconjunctivitis nPCR is superior to viral culture and tear film is an adequate sample that is easier to collect, causing the patient less discomfort.