The physicochemical properties of two serologically distinct equine rhinoviruses have been examined. Each virus sedimented at approximately 160S but co-centrifugation of the two viruses in a sucrose gradient revealed a small difference in their sedimentation coefficients. The two viruses also have different buoyant densities in cesium chloride. The equine rhinovirus type 1 equilibrated as a sharp peak at 1.45 g/ml whereas the type 2 virus equilibrated as a heterogeneous band with a peak at 1.44 g/ml but ranging in density from 1.41 to 1.45 g/ml. The relative sedimentation coefficients of the two virus RNAs were 35S for rhinovirus 1 and 37S for rhinovirus 2. A limited number of base composition analyses also showed differences between the two virus RNAs. The polypeptide profile of each serotype in polyacrylamide gels was generally similar to those of other picornaviruses but the two serotypes could be distinguished readily from each other.