The influenza neuraminidase (NA) inhibitors peramivir, oseltamivir, and zanamivir are potent inhibitors of NAs from both influenza A and B strains. In general, these inhibitors are slow, tight binders of NA, exhibiting time-dependent inhibition. A mutant of influenza virus B/Yamagata/16/88 which was resistant to peramivir was generated by passage of the virus in tissue culture, in the presence of increasing concentrations (0.1–120 μM over 15 passages) of the compound. Whereas the wild type (WT) virus was inhibited by peramivir with an EC 50 value of 0.10 μM, virus isolated at passages 3 and 15 displayed EC 50 values of 10 and >50 μM, respectively. Passage 3 virus contained 3 hemagglutinin (HA) mutations, but no NA mutation. Passage 15 (P15R) virus contained an additional 3 HA mutations, plus the NA mutation His273Tyr. The mechanism of inhibition of WT and P15R NA by peramivir was examined in enzyme assays. The WT and P15R NAs displayed IC 50 values of 8.4±0.4 and 127±16 nM, respectively, for peramivir. Peramivir inhibited the WT enzyme in a time-dependent fashion, with a K i value of 0.066±0.002 nM. In contrast, the P15R enzyme did not display the property of slow binding and was inhibited competitively with a K i value of 4.69±0.44 nM. Molecular modeling suggested that His273 was relatively distant from peramivir (>5 Å) in the NA active site, but that Tyr273 introduced a repulsive interaction between the enzyme and inhibitor, which may have been responsible for peramivir resistance.