Posttranscriptional controls modulate the expression of several human cytomegalovirus genes. Previous studies have shown that one cytomegalovirus gene transcript leader contains AUG codons which inhibit translation of a downstream reading frame. However, two other cytomegalovirus gene transcript leaders of similar structure do not inhibit translation. We have extended these studies to the analysis of the structural glycoprotein gp48, whose predominant transcript contains three upstream AUG codons. The 5' leader of this transcript strongly inhibits downstream translation in fibroblasts. Analyses of deletions and point mutations identify the second upstream AUG codon as an essential component of the inhibitory signal. Other leader sequences, but neither the first nor the third AUG codon, are also required. Intriguingly, the inhibitory signal appears also to depend on the amino acid coding information of the short reading frame associated with the second AUG codon. Insights derived from these studies are germane to understanding the translational regulation of other viral and cellular genes of similar structure.