An inhibitor against type 1 poliovirus associated with the gamma globulin of certain horse sera was investigated. Addition of inhibitory horse serum (IHS) at a concentration of 2 1 2 % in the overlay medium caused marked reduction in plaque size and a delay of 24–48 hours in plaque development. Plaque counts were unaffected at this serum concentration, but were reduced by 50 % with 5 % serum. Inhibitor-resistant particles capable of forming normal-sized plaques in the presence of IHS were found in a plaque-purified pool of type 1 poliovirus (Mahoney strain). Based on plaque size, inhibitor-sensitive and -resistant particles could be differentiated in naturally occurring type 1 virus in stools of patients with paralytic poliomyelitis. The inhibitor had no effect on plaque development of type 2 and 3 polioviruses, Coxsackie A9, vaccinia, or encephalomyocarditis virus (EMC). The inhibitor differs from the previously described bovine serum inhibitor in not having neutralizing ability; no evidence was found that it is viral antibody. Growth-curve experiments showed that the inhibitor reduces rate of adsorption of virus to susceptible cells and delays release of virus from infected cells.