The emergence of virulent avian influenza viruses in poultry is unpredictable. To gain insight into the mechanism for this event, we sought to identify the molecular changes in virulent mutants that occur during replication in 14-day-old embryonated chicken eggs. After three passages in 14-day-old eggs, avirulent H5 viruses with the K/R-K-K/T-R sequence at the hemagglutinin (HA) cleavage site became virulent in chickens, concomitantly acquiring high HA cleavability, whereas those with the R-E-T-R sequence did not. None of the test viruses converted to a virulent phenotype when passaged in 10-day-old eggs. Nucleotide sequence analysis indicated that the virulent mutants either lost a glycosylation site near the HA cleavage site or acquired an additional arginine at the latter. Avirulent viruses that became virulent after passage in older eggs included an H5N2 avian strain with the R-K-T-R sequence that was isolated in 1993, indicating that viruses with this sequence motif, which are currently circulating in bird populations, should be considered potentially virulent. Failure to generate virulent mutants from viruses with R-E-T-R at the HA cleavage site underscores the pathogenic heterogeneity among avian influenza viruses.