Influenza A viruses of the H5N1 A/HongKong/483/97 (H5N1), A/Viet Nam/1203/04 (H5N1), A/Anhui/1/2005 (H5N1), and A/ Indonesia/5/2005 (H5N1) were grown in the allantoic cavity of 10day-old embryonated hen eggs at 37uC for 26 to 40 h. Allantoic fluid pooled from multiple eggs was clarified by centrifugation and frozen in aliquots at 270uC. The 50% egg infectious dose (EID50) for each virus stock was calculated by the method of Reed and Muench following serial titration in eggs. All experiments with HPAI viruses were conducted under Biosafety Level 3 containment.