To determine whether other avian, but not human, influenza viruses display temperature dependent phenotypes, we performed multi-step growth curves with more human H3N2 isolates (A/ Eng/26/99 and A/Udorn/307/72) and A/Dk/Sing/97, an avian isolate of different subtype (H5N3). Growth of both humanderived influenza viruses tested, A/Eng/26/99 (H3N2) and A/ Udorn/307/72 (H3N2), was not significantly different between 32/33uC and 37uC ( Figure 3A and 3B). Indeed, these two additional human influenza virus strains showed even less difference in titer between temperatures than was determined for A/Victoria/3/75. Assessment of growth of avian influenza virus, A/Dk/Sing/97 (H5N3), over a 48 hr time course at 37uC showed similar growth kinetics to that of A/Eng/26/99 (H3N2), reaching titers of 7610 5 pfu/ml and 1.6610 6 pfu/ml, respectively ( Figure 3A and 3C). In contrast, at 32uC, A/Dk/Sing/97 (H5N3) failed to grow at all ( Figure 3C ). Clearly, the restriction of A/Dk/Sing/97 at 32uC compared to 37uC was an even more striking phenotype than A/ Duck/Eng/62. As the avian influenza virus strains used in this study were selected at random, with no selection for a temperature-dependent phenotype, we propose that low temperature restriction of avian influenza viruses, but not human influenza viruses, may be broadly characteristic of avian influenza viruses. The extent of restriction, however, may be variable between different virus strains.