In 1970, a great outbreak of hand‐foot‐and‐mouth disease (HFMD) due to Coxsackie A16 virus (Cox A16) occurred throughout Japan. When serologic relationship between the viruses isolated during the epidemic and the prototype strain of the serotype was examined by the tube neutralization test, the crude new isolates were found to be poorly neutralized by both an antiserum against the prototype strain and those against the isolates, although they were neutralized significantly in the plaque reduction test. However, about 2% of virus in crude suspensions of the isolates remained unneutralized even in the plaque reduction test and this fraction could be eliminated by filtering the virus materials through a 100 mμ Millipore filter. Therefore, the difficulty in neutralization of the isolates by the tube method could be accounted for by the presence of aggregated viruses. Even when filtered viruses were used, the reciprocal neutralization kinetic studies revealed a significant serological difference between the isolates and the prototype strain. Such serological properties of the isolates were not influenced by the cell types used for virus isolation or passages. All the results suggest that the Cox A16 isolates from the epidemic of HFMD in Japan, 1970, are serologically different from the prototype strain.