The replication of murine leukemia virus (MuLV) requires the capture of a cellular tRNAPro as a primer for reverse transcription. To further study the specificity of primer selection, we have utilized a defective MuLV in which the primer-binding site (PBS) has been altered to be complementary to a nonmammalian tRNA, yeast tRNAPhe. Infectivity of the defective MuLV is dependent upon co-expression of yeast tRNAPhe in the cell. Defective MuLV genomes have been constructed in which the PBS was altered to be complementary to tRNAPhe that also encoded the cDNA for tRNA Phe. Transfection of these defective proviral genomes into cells resulted in the production of infectious MuLV as determined by a single-round assay. The amount of infectious virus produced using this complementation system, though, was approximately 6-fold lower than that produced following transfection of defective proviral genomes with a wild-type PBS complementary to tRNAPro. The lower infectivity was not due to reduced expression of tRNAPhe in the transfected cells as compared to endogenous tRNA Pro or tRNALys,3. Serial passage of the MuLV genome with a PBS complementary to tRNAPhe that encoded tRNAPhe resulted in amplification of the virus. Using this rescue system, we have passaged the virus for four serial passages, after which time a revertant genome in which the PBS was altered to be complementary to tRNAGln was detected that grew to high titers following subsequent serial passage. The results of these studies suggest that MuLV has preferences for the tRNA primer used in reverse transcription and are discussed with respect to the mechanism of primer selection. © 2004 Elsevier Inc. All rights reserved.