We evaluated the effect of ganciclovir on in vitro immune responses by lymphocytes from healthy individuals. At clinically relevant concentrations, ganciclovir inhibited proliferation responses to cytomegalovirus antigen and to phytohemagglutinin by >50% but had no effect on production of interleukin-2 or γ interferon to these stimuli or on cytotoxicity against cytomegalovirus-infected, HLA-mismatched target cells. These results showed that ganciclovir inhibits lymphocyte functions for which lymphocyte proliferation is required but does not affect lymphocyte responses that do not require DNA synthesis. Studies of the effect on the immune responses in patients receiving ganciclovir will be needed to determine in vivo correlates of these findings.