D,L-alpha-Difluoromethylornithine (DFMO) is an inhibitor of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway. Exposure of human foreskin fibroblast cells to DFMO before their infection with human strains of cytomegalovirus (CMV) resulted in a reduction in the amount of infectious virus produced. A 3-day exposure to the drug was required to elicit maximal antiviral effect. Cells exposed to DFMO at the time of infection produced normal amounts of infectious virus. Preexposure to the drug for 1, 2, or 3 days before infection resulted in at least 10-, 100-, or 1,000-fold decreases, respectively, in the amount of infectious virus produced. This decrease paralleled the loss of intracellular spermidine and was partially spared by the addition of exogenous putrescine, spermidine, or spermine (10 microM). When added 3 days before infection, DFMO depressed production of herpes simplex virus and simian CMV, as well as wild-type and laboratory prototype strains of human CMV. Although some antiviral effect was observed at a drug concentration of 1 mM, 10 mM gave a stronger effect and was the amount routinely used. At 30 mM DFMO, growth of noninfected cells was slowed but not arrested. Studies to investigate the level at which DFMO interferes with CMV replication showed that DFMO-treated, infected cells (i) exhibit a typical CMV-specific cytopathic effect, (ii) synthesize both viral proteins and viral DNA, (iii) contain at least some capsid forms, and (iv) shed greatly reduced amounts of virus particles into the growth medium. Since CMV virions, like those of herpes simplex virus, contain the polyamines spermidine and spermine, and since DFMO essentially eliminates the pool of intracellular spermidine, the possibility is suggested that this drug may exert its antiviral effect by interfering with virus assembly, perhaps at the level of DNA packaging or capsid envelopment or both.