Reports of human cases of infection with avian influenza A(H5N1) virus have received increased public attention because of the potential for the emergence of a pandemic strain. In the end of 2005 and the beginning of 2006, avian influenza A(H5N1) virus caused outbreaks among domestic poultry and was isolated from wild swans in many European countries, including Bulgaria. Between January and March 2006, samples were collected from 26 patients who had been in close contact with ill or dead birds and developed a subsequent respiratory illness. The testing took place at the National Laboratory of Influenza in Sofia. Specific A(H5N1) assays were applied for screening (Sacace RT-PCR and real-time kit). Avian flu A(H5N1) virus was not detected in any of the patients tested. In three patients, human subtype A(H1N1) influenza virus, identifiable by RT-PCR was isolated and further characterized by hemagglutination inhibition test (HIT). The reliability of the molecular assays used in this investigation was demonstrated in an International Quality Control for Human and Avian A(H5N1) Influenza performed later in 2006 by INSTAND (Society for Promotion of Quality Assurance in the Medical Laboratories), Germany.