Ca2+ increases the initial rate of activity of sialidase from influenza virus (A/Tokyo/3/67). Increasing ionic strength also activates influenza virus sialidase. When ionic strength is controlled, smaller but still significant Ca2+ effects are observed, with Vmax/Km increased from 0.8.10(5) to 1.4.10(5) M-1 s-1 and Vmax increased from 6.3 to 9.5 s-1 by saturating Ca2+. The Ki of the competitive inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid was decreased from 2.7.10(-6) to 1.15.10(-6) M after the addition of saturating Ca2+. The data show that Ca2+ exerts a specific effect on Vmax/Km, leading to an increased rate of interaction of substrate with the enzyme. The Kd-app for the Ca2(+)-sialidase complex is 2 mM. Except for Mg2+ which behaves similarly to Ca2+, other mono- and divalent cations have little specific effect on sialidase kinetics. Sequence analysis of a range of subtypes of sialidases from influenza virus supports the proposal that Ca2+ binds at the subunit interface transmitting a conformational change to the enzyme active site. Ca2+ activation may have a physiological role in switching on sialidase activity during the release of newly synthesised virions from the host cell surface.