A procedure for labeling influenza viruses with alkylamine-modified horseradish peroxidase (HRP) was developed to study virus receptor specificities in a virus overlay assay. Six alkylamine derivatives of HRP were prepared by the periodate oxidation method. Human influenza virus A/PR/8/34 (H1N1) was labeled with the alkylamine derivatives, and a variety of parameters for labeling were tested in a fetuin-immobilized microplate solid-phase assay. The labeling efficiency of influenza virus with the derivatives of HRP was affected by the alkyl chain length of the derivatives. The most effective derivative for labeling the virus being tested was octylamine-modified HRP (octyl-HRP). The labeling profile of A/PR/8/34 by octyl(C8H17)-HRP was analyzed b sucrose density gradient centrifugation. The labeling had no effect on viral hemagglutinating activity and fetuin binding specificity. The specific binding of the HRP-labeled human influenza viruses (A/PR/8/34 and B/Lee/40) to glycolipids was demonstrated by a virus overlay assay. In this virus-HRP-labeled system, the virions which bound to gangliosides developed on a silica gel thin-layer plate were detected simply and rapidly.