Forty-two influenza A and 23 influenza B isolates collected from untreated subjects during the 1999–2000 influenza season in Canada were tested for their susceptibility to three neuraminidase inhibitors (zanamivir, oseltamivir carboxylate and RWJ-270201 or BCX-1812) using a chemiluminescent neuraminidase assay. Influenza B isolates were less susceptible than A viruses to all tested drugs. RWJ-270201 was the most potent drug against both influenza A(H3N2) (mean IC50: 0.60 nM) and B (mean IC50: 0.87 nM) viruses. Oseltamivir carboxylate was more active than zanamivir for influenza A(H3N2) isolates (mean IC50: 0.73 vs. 2.09 nM) whereas it was less potent against B viruses (mean IC50: 11.53 vs. 4.15 nM).