Rigorous investigation of many functions encoded by cytomegaloviruses (CMVs) requires analysis in the context of virus-host interactions. To facilitate the construction of rhesus CMV (RhCMV) mutants for in vivo studies, a bacterial artificial chromosome (BAC) containing an enhanced green fluorescent protein (EGFP) cassette was engineered into the intergenic region between unique short 1 (US1) and US2 of the full-length viral genome by Cre/lox-mediated recombination. Infectious virions were recovered from rhesus fibroblasts transfected with pRhCMV/BAC-EGFP. However, peak virus yields of cells infected with reconstituted progeny were 10-fold lower than wild-type RhCMV, suggesting that inclusion of the 9-kb BAC sequence impeded viral replication. Accordingly, pRhCMV/BAC-EGFP was further modified to enable efficient excision of the BAC vector from the viral genome after transfection into mammalian cells. Allelic exchange was performed in bacteria to substitute the cre recombinase gene for egfp. Transfection of rhesus fibroblasts with pRhCMV/BAC-Cre resulted in a pure progeny population lacking the vector backbone without the need of further manipulation. The genomic structure of the BAC-reconstituted virus, RhCMV-loxP(r), was identical to that of wild-type RhCMV except for the residual loxP site. The presence of the loxP sequence did not alter the expression profiles of neighboring open reading frames. In addition, RhCMV-loxP(r) replicated with wild-type kinetics both in tissue culture and seronegative immunocompetent macaques. Restriction analysis of the viral genome present within individual BAC clones and virions revealed that (i) RhCMV exhibits a simple genome structure and that (ii) there is a variable number of a 750-bp iterative sequence present at the S terminus.