Nonsense-mediated mRNA decay (NMD) specifically targets mRNAs with premature translation termination codons for rapid degradation. NMD is a highly conserved translation-dependent mRNA decay pathway, and its core Upf factors are thought to be recruited to prematurely terminating mRNP complexes, possibly through the release factors that orchestrate translation termination. Upf1 is the central regulator of NMD and recent studies have challenged the notion that this protein is specifically targeted to aberrant, nonsense-containing mRNAs. Rather, it has been proposed that Upf1 binds to most mRNAs in a translation-independent manner. In this thesis, I investigated the nature of Upf1 association with its substrates in the yeast Saccharomyces cerevisiae. Using biochemical and genetic approaches, the basis for Upf1 interaction with ribosomes was evaluated to determine the specificity of Upf1 association with ribosomes, and the extent to which such binding is dependent on prior association of Upf1’s interacting partners. I discovered that Upf1 is specifically associated with Rps26 of the 40S ribosomal subunit, and that this association requires the N-terminal Upf1 CH domain. In addition, using selective ribosome profiling, I investigated when during translation Upf1 associates with ribosomes and showed that Upf1 binding was not limited to polyribosomes that were engaged in translating NMD substrate mRNAs. Rather, Upf1 associated with translating ribosomes on most mRNAs, binding preferentially as ribosomes approached the 3’ ends of open reading frames. Collectively, these studies provide new mechanistic insights into NMD and the dynamics of Upf1 during translation.