BACKGROUND Highly pathogenic avian influenza virus (HPAIV) H5N1 infections in felids have been reported in several countries. Feeding on infected birds has been suggested as potential source of infection. OBJECTIVE The study aimed to verify gastrointestinal infection as possible portal of entry for HPAIV H5N1 in cats. METHODS Four cats were infected oculo-nasopharyngeally with 10⁶ 50% egg infectious dose (EID(50) ) of HPAIV H5N1 A/cat/Germany/R606/2006. Two cats were infected intravenously with 10⁶ EID(50) and two cats were inoculated orally with 10⁷ EID(50) HPAIV embedded in gelatine capsules to mimic gastrointestinal exposure and to avoid virus contact to oropharyngeal or respiratory tissues. Cats were monitored for 6 days by physical examination, virus excretion, and peripheral blood lymphocyte counts. Blood chemical parameters (including AST, ALT, CPK, and TBIL) and viral excretion using pharyngeal and rectal swabs were analyzed. RESULTS Infected cats showed elevated body temperature up to 41·3°C starting from day 1 or 2 p.i. All infected cats excreted virus in pharyngeal swabs within 2 days p.i. co-inciding with the development of clinical signs (anorexia, depression, and labored breathing) irrespective of the infection route. Virus dissemination occurred through cell-free and cell-associated viremia. Infected cats developed lymphopenia, hepatic necrosis, pneumonia, and significantly elevated levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine phosphokinase (CPK), and TBIL. CONCLUSIONS The experiments show that the gastrointestinal tract can serve as portal for the entry of HPAIV H5N1 into cats. Infection routes used did not influence viral tissue tropism and course of disease.