Several animal viruses are known to contain significant amounts of polyamines but so far the function of these viral components is poorly understood. In this study the role of polyamines in the replication of two different types of viruses, herpes simplex virus type 2 and Semliki Forest virus (SFV) has been investigated. Purified SFV was found to contain fairly small amounts of polyamines, sufficient to neutralize only about 3% of viral nucleic acid phosphate, i.e., 1/20 of that found in herpes simplex virus. The production of both viruses was, however, markedly inhibited in BHK21 cells depleted of polyamines by treatment with alpha-difluoromethylornithine, a specific inhibitor of polyamine synthesis. This inhibition was reversed by putrescine, spermidine and spermine, and at least partly reversed by several other diamines and polyamine homologs. The activity of viral RNA polymerase induced by SFV infection was markedly reduced in polyamine-depleted cells but increased rapidly after addition of spermidine to the culture medium. It appears that the inhibition of virus production in polyamine-depleted cells is due in part to malfunction of protein synthetic machinery of the host cell. The possibility that other steps in virus synthesis and assembly are affected by polyamine deficiency is currently being investigated.