Rifampin at a concentration of 150 μg/ml interfered with the replication of Davis, Kerr, and AD 169 strains of cytomegalovirus in Wi-38 cells. The replication of herpes-simplex virus (types 1 and 2) and Sindbis virus in the same cells was unaffected by a similar concentration of the antibiotic. Direct inactivation of the virus and interference with adsorption and release of the virus were excluded as possible mechanisms for the inhibitory effect.