The kinetics of rhesus cytomegalovirus (RhCMV) infection were compared in primary and telomerase-immortalized (Telo-) rhesus fibroblasts (RF). Equivalent viral titers were achieved with both cell types. However, the production of infectious virions was slightly faster and plaque size was larger in Telo-RF, compared with primary cells. Comparable RhCMV growth curves and viral susceptibility were observed using Telo-RF passaged for different time periods in culture, whereas the ability of primary cells to support robust RhCMV replication declined as the cells approached senescence. Analysis of cell growth kinetics suggested that the rate of RhCMV replication was directly related to the rate of cell proliferation. RT-PCR analysis of representative RhCMV genes demonstrated that the presence of telomerase did not alter the temporal profile of RhCMV gene expression. In addition, Telo-RF cells were observed to have a significantly increased efficiency of transfection with cationic lipids, compared with primary RF. These results demonstrated that Telo-RF represents a stable, permissive cell line for RhCMV infection, facilitating standardization of in vitro assays for this important non-human primate CMV. The ease of transfection will enable molecular analyses and the generation of complementing cell lines for the propagation of defective RhCMV variants.