The Saccharomyces cerevisiae CPAI mRNA 5'-leader contains an evolutionarily conserved 25-codon upstream open reading frame (uORF), which genetic studies had suggested to encode a peptide involved in translational regulation in response to arginine (Arg). Using a cell-free translation system, studies with the CPAI homolog in Neurospora crassa, arg-2, showed that the arg-2 uORF-encoded peptide, the Arg attenuator peptide (AAP), functioned to stall ribosomes at the uORF termination codon in response to Arg. To investigate CPAl AAP function, this study utilized S. cerevisiae and N. crassa cell-free translation systems to demonstrate that the CPAI AAP functions as its N. crassa counterpart to stall ribosomes at its uORF termination codon in response to Arg, resulting in reduced levels of ribosomes at a downstream reporter start codon. The charge status of Arg-tRNA and the termination of AAP translation did not appear critical for S. cerevisiae AAP function to stall ribosomes. Analyses of ribosomes at the CPAl uORF and at the uORFs present in GCN4 mRNA indicated that these uORFs control translation in fundamentally different ways. Whereas the CPAl uORF regulates ribosomes that scan past the uORF, the GCN4 uOWs regulate ribosomes that reinitiate translation. CPAl uORF analyses using an in vivo reporter system with wild-type and nonsense-mediated mRNA decay (NMD)-deficient strains indicated that uOW-mediated translational control regulated CPAl mRNA levels by affecting transcript susceptibility to NMD. These studies of the CPAl uORF provide a novel example of regulated eukaryotic gene expression in which the synthesis of a nascent peptide controls translation and mRNA stability.