The effect of 2,3-didehydro-2,4-dideoxy-4-guanidino-N-acetyl-D-neuraminic acid (4-guanidino-Neu5Ac2en) on the sialidases from influenza virus reassortant X31 (which contains the sialidase from A/Aichi/2/68) and influenza virus B/Beijing/1/87 has been investigated. We find that 4-guanidino-Neu5Ac2en is a slow-binding inhibitor of both influenza A and influenza B virus sialidase, and that association and dissociation rate constants are almost identical for both enzymes. Furthermore, values for these rate constants are independent of whether purified enzyme or detergent-treated virus is used in the assays.