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Cytoplasmic dynein-1 (dynein) is a microtubule-based motor protein that is essential for maintaining proper spatial and temporal organization of the cellular interior during both interphase and cell division. To do so, dynein carries out diverse functions including transport of intracellular cargos, organelle anchoring, mitotic spindle assembly and nuclear migration. To carry out these diverse functions, dynein is regulated by multiple factors including Lis1, dynactin and activating adaptors. In this thesis, I present interdisciplinary work investigating the mechanisms of regulation of dynein. In chapter 2, a combination of biochemistry, single-molecule assays and cryo-electron microscopy was used to investigate how yeast dynein is regulated by Lis1. Previous work showed that Lis1 binds directly to dynein’s AAA+ motor domain and slows its motility by causing dynein to tightly bind microtubules. The work presented here revealed that in addition to this role, Lis1 can also induce dynein to weakly bind microtubules. The mode of regulation used by Lis1 is determined by the nucleotide state at dynein’s third AAA domain and the stoichiometry of the Lis1-dynein interaction. Both modes of Lis1 regulation of dynein are required for dynein to position the mitotic spindle in yeast cells. In chapter 3, the mechanism of regulation of human dynein by Lis1 is investigated. Human dynein requires dynactin and an activating adaptor for directional long-distance movement on microtubules. The work presented here shows that Lis1 promotes the formation of a maximally activated dynein complex consisting of dynein, dynactin and an activating adaptor. Finally, in chapter 4, the identification of the dynein protein interactome is described. Two novel dynein activating adaptors, ninein and ninein-like, were discovered and shown to activate dynein/dynactin motility in single-molecule assays. Overall, the work presented here reveals novel insights into complex regulation of dynein., Biophysics