The regulation of human cytomegalovirus gene expression depends on both transcriptional and post-transcriptional controls. Previous studies revealed that either of two AUG codons contained in the 5'leader of a beta gene (2.7 beta) transcript inhibited translation of a downstream reading frame. We investigated the regulatory effects of 5' leader sequences from the cytomegalovirus DNA polymerase and pp150 genes, each of which also contains upstream AUG codons. Surprisingly, these two leaders did not affect expression of the downstream open reading frame. Detailed analyses were carried out to examine the role of the AUG codons within the pp150 leader. These upstream AUG codons allowed efficient downstream translation, despite the predictions of the scanning model of eukaryotic translation. Further studies of the 2.7 beta leader revealed that an upstream AUG codon, although necessary, was not sufficient to inhibit downstream translation. These results reveal that translational inhibition by CMV transcript leaders requires an AUG codon and additional leader sequences.