The simian virus 40 (SV40)-yielding variants of the adenovirus type 2 (Ad.2)-SV40 hybrid (Ad.2(++)) population were studied by means of fixed-angle equilibrium density gradient centrifugation in cesium chloride. The hybrid virions of the Ad.2(++) high-efficiency yielder population banded at densities of 0.004 g/cm(3) lighter than the nonhybrid Ad.2 virions. The degree of separation of the hybrid particles was sufficient to permit greater than 100-fold relative purification by two cycles of centrifugation. Hybrid particles that produce adenovirus plaques in African green monkey kidney cells by two-hit kinetics (one-hit kinetics when assayed on lawns of nonhybrid adenovirus) were not separable from the particles that yield SV40 virus. The hybrid particle in the Ad.2(++) low-efficiency yielder population was not separable from the nonhybrid Ad.2 virions.