Highly pathogenic avian influenza viruses of the H5N1 subtype continue to circulate in poultry in Asia, Africa, and the Middle East. Recently, outbreaks of novel reassortant H5 viruses have also occurred in North America. Although the number of human infections with highly pathogenic H5N1 influenza viruses continues to rise, these viruses remain unable to efficiently transmit between humans. However, we and others have identified H5 viruses capable of respiratory droplet transmission in ferrets. Two experimentally introduced mutations in the viral hemagglutinin (HA) receptor-binding domain conferred binding to human-type receptors but reduced HA stability. Compensatory mutations in HA (acquired during virus replication in ferrets) were essential to restore HA stability. These stabilizing mutations in HA also affected the pH at which HA undergoes an irreversible switch to its fusogenic form in host endosomes, a crucial step for virus infectivity. To identify additional stabilizing mutations in an H5 HA, we subjected a virus library possessing random mutations in the ectodomain of an H5 HA (altered to bind human-type receptors) to three rounds of treatment at 50°C. We isolated several mutants that maintained their human-type receptor-binding preference but acquired an appreciable increase in heat stability and underwent membrane fusion at a lower pH; collectively, these properties may aid H5 virus respiratory droplet transmission in mammals. OBJECTIVE We have identified mutations in HA that increase its heat stability and affect the pH that triggers an irreversible conformational change (a prerequisite for virus infectivity). These mutations were identified in the genetic background of an H5 HA protein that was mutated to bind to human cells. The ability to bind to human-type receptors, together with physical stability and an altered pH threshold for HA conformational change, may facilitate avian influenza virus transmission via respiratory droplets in mammals.