Cell culture systems were developed for rapid antiviral drug screening, using murine cytomegalovirus (MCMV) as an alternative to the slower growing human CMV. Since previous assay methods with MCMV employed mouse embryo fibroblasts (MEF cells), which are labor intensive to prepare and die off after 3-4 passages from primary culture, identification of virus-susceptible continuous cell lines was desirable. Three cell lines were found useful for assaying MCMV: C127I, SC-1, and 3T3. The antiviral agents acyclovir, ganciclovir, 5-fluoroarabinofuranosylcytosine, and 2'-fluoro-2'-deoxy-5-iodoarabinofuranosylcytosine were evaluated in the 3 continuous cell lines and in MEF cells. The 50% virus- or cell-inhibitory concentration values determined for each compound did not vary much from cell to cell. MEF cells were 10-fold more sensitive than the other cell lines to quantify virus from mouse organs, however. Virus propagated in 3T3 and SC-1 cells were as virulent to mice as salivary gland virus, whereas virus from MEF and C127I cells was more attenuated. Overall, C127I cells were judged to be the best for large scale antiviral screening in vitro, but MEF was the cell type of choice for titration of viruses from mouse organs and tissues.