HeLa cells were made strictly dependent upon polyamine by growth in the presence of alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase. Under these conditions, the specific activity of the cellular polyamine pools eventually equilibrated to that of exogenously supplied [14C]putrescine; however, the process was very slow, requiring half-equilibration times of about 16 h for spermidine and 28 for spermine. Thus, the distribution of radioactivity in individual polyamines became a valid measure of polyamine content only after a continuous 4-day incorporation period. When propagated in polyamine-labeled cells, two picornaviruses were found to incorporate substantially different amounts of polyamine: about 0.6% of the cell pool for human rhinovirus 14 but only 0.04% for poliovirus. This content of polyamine was sufficient to neutralize nearly 27% of the negative charge of the RNA in human rhinovirus 14 but only 1.6% in poliovirus.