A simple polymerase chain reaction (PCR)-based procedure was developed for the detection of avian infectious laryngotracheitis virus (ILTV) in chicken trachea, chorio-allantoic membrane (CAM), infected hepatoma cells and infectious cell culture supernatant. Samples were prepared by dilution in distilled water. After boiling and low speed centrifugation, samples were used for PCR analysis with two primers without special labeling. The PCR analysis for ILT virus could be completed in less than 8 h. Standard agarose gel electrophoretic analysis of the PCR products revealed a prominent band of 300 base-pairs in samples from ILTV-infected specimens, but not from specimens containing Newcastle disease virus, infectious bronchitis virus, avian adenovirus, fowlpox virus, Pachecoz or Marek's disease virus. One single ILTV infected cell or 10 plaque forming units of ILTV could be detected with this procedure. The procedure can be used for the identification of ILTV and the differentiation of ILTV from other avian respiratory tract infectants.