Cytochrome P450 46A1 (CYP46A1) is a key enzyme responsible for cholesterol elimination from the brain. This P450 can interact with different steroid substrates and protein redox partners. We utilized hydrogen-deuterium (H-D) exchange mass spectrometry for investigating CYP46A1-ligand interactions. First, we tested the applicability of the H-D exchange methodology and assessed the amide proton exchange in substrate-free and cholesterol-sulfate-bound P450. The results showed good correspondence to the available crystal structures and prompted investigation of the CYP46A1 interactions with the two steroid substrates cholesterol and 24S-hydroxycholesterol and the protein redox partner adrenodoxin (Adx). Compared to substrate-free P450, four peptides in cholesterol-bound CYP46A1 (65-80, 109-116, 151-164, and 351-361) and eight peptides in 24S-hydroxycholesterol-bound enzyme (50-64, 65-80, 109-116, 117-125, 129-143, 151-164, 260-270, and 364-373) showed altered deuterium incorporation. Most of these peptides constitute the enzyme active site, whereas the 351-361 peptide is from the region putatively interacting with the redox partner Adx. This also defines the proximal (presumably water) channel that opens in CYP46A1 upon substrate binding. Reciprocal studies of Adx binding to substrate-free and cholesterol-sulfate-bound CYP46A1 revealed changes in the deuteration of the Adx-binding site 144-150 and 351-361 peptides, active site 225-239 and 301-313 peptides, and in the 265-276 peptide, whose functional role is not yet known. The data obtained provide structural insights into how substrate and redox partner binding are coordinated and linked to the hydration of the enzyme active site.