Human cytomegalovirus (HCMV) infection of low serum-arrested confluent whole human embryo (Flow 5000) cells markedly stimulated ornithine decarboxylase (ODC) activity. Increased ODC activity was apparent by 12 h post-infection. The capacity of HCMV to stimulate ODC was: (1) dependent upon multiplicity of infection; (2) eliminated when the virus was neutralized with specific antiserum; and (3) sensitive to ultraviolet irradiation. Virus-mediated induction, in contrast to high serum induction of ODC, was not subject to inhibition by polyamines added to the growth medium. Phosphonoacetic acid (PAA) which blocks HCMV replication by inhibiting the activity of HCMV-specific DNA polymerase and which does not prevent HCMV induced stimulation of cell DNA synthesis, reversibly inhibited HCMV-induced stimulation of ODC activity by 74%. Studies with PAA indicated that HCMV-induced stimulation of ODC activity is independent of cell DNA synthesis and that the mechanism regulating virus-induced stimulation may be related to the HCMV-specific DNA polymerase.