The HIV-1 accessory protein Nef is N-terminally myristoylated, and this post-translational modification is essential for Nef function in AIDS progression. Transfer of a myristate group from myristoyl coenzyme A to Nef occurs cotranslationally and is catalyzed by human N-myristoyltransferase 1 (NMT). To investigate the conformational effects of myristoylation on Nef structure as well as to probe the nature of the Nef:NMT complex, we investigated various forms of Nef with hydrogen exchange mass spectrometry. Conformational changes in Nef were not detected as a result of myristoylation, and NMT had no effect on deuterium uptake by Nef in a myrNef:NMT complex. However, myrNef binding did have an effect on NMT deuterium uptake. Major HX differences in NMT were primarily located around the active site, with more subtle differences, at the longer time points, across the structure. At the shortest time point, significant differences between the two states were observed in two regions which interact strongly with the phosphate groups of coenzyme A. On the basis of our results, we propose a model of the Nef:NMT complex in which only the myristoyl moiety holds the two proteins together in complex and speculate that perhaps NMT chaperones Nef to the membrane and thereby protects the myristic acid group from the cytosol rather than Nef operating through a myristoyl switch mechanism.