A short upstream open reading frame (uORF2) in the human cytomegalovirus (CMV) gpUL4 (gp48) transcript leader is conserved among CMV strains and inhibits translation of a downstream cistron. Remarkably, this inhibitory effect depends on the amino acid coding information of uORF2, at least in transient transfection assays in diploid human fibroblasts. Using retroviral vectors, we now report that the gp48 leader inhibits downstream translation in multiple additional cell types, even when expressed from a stably integrated gene, and on a transcript containing an additional kilobase of complex leader sequences. The magnitude of inhibition can be augmented approximately 3- to 10-fold by replacing the context of nucleotides flanking the wild-type initiation codon of uORF2 with an optimal context, suggesting that leaky scanning past the wild-type AUG codon accounts for translation of the downstream cistron. Using an in vivo mutagenesis protocol that relies on reverse transcriptase infidelity, we isolated mutants in which the inhibitory effect of the gp48 leader was inactivated as a result of alterations in the coding information of uORF2. These studies demonstrate that, independent of the cell type or expression system used, CMV gp48 uORF2 is a potent translational inhibitory element.