The neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) of the influenza virus recombinant strain (HON2) was solubilized with detergents and isolated by affinity chromatography. The neuraminidase could be purified to a single high molecular weight glycoprotein when assayed under non-reducing conditions on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme showed an increase in specific activity from 2.46 to 189 micronM N-acetylneuraminic acid released per min per mg protein and the recovery represented 123% of the activity of intact virus particles. The enzyme could be purified from laboratory preparations of virus or from outdated influenza virus vaccine. Viral neuraminidases purified by this technique were stable at pH 6.0 for several hours.