Equine herpesvirus-1 (EHV-1) causes respiratory disease, neonatal death, abortion and neurologic disease. The main purpose of this study was to identify viral antigen in respiratory tract samples by immunoperoxidase staining. Six pony foals were selected on the basis of demonstrating seronegativity to EHV-1 by virus neutralization and housed in isolation. They were infected experimentally by administering EHV-1 nebulized ultrasonically through a face mask. Successful infection was clinically apparent as each of the foals had febrile responses, nasal discharge, and enlarged submandibular lymph nodes. Sporadic coughing was also heard. EHV-1 was isolated from nasopharyngeal swabs of 4/6 ponies and seroconversion was demonstrated in all foals. Bronchoscopic examination of the large airways revealed hyperemia. The incidence of recovery of Actinobacillus suis from nasopharyngeal swabs increased initially, with recovery of Streptococcus zooepidemicus isolates predominating at 3 wk post-infection. Cytology brushes were used to sequentially sample the respiratory tract of the infected ponies at the nasopharynx, mid-trachea and the mainstem bronchus. Bronchoalveolar lavage provided lung cells. Immunocytochemistry techniques were applied to both types of samples to locate EHV-1 antigen. Indirect immunoperoxidase staining of samples utilizing monoclonal antibodies specific for EHV-1 demonstrated viral antigen associated with cellular debris, primarily in the nasopharyngeal samples on days 3-9 post-infection.