The influenza A virus M2 protein is a pH-gated and amantadine-inhibited proton channel important for the virus life cycle Proton conduction by M2 is known to involve water; however direct experimental evidence of M2-water interaction is scarce Using H-1 spin diffusion solid-state NMR, we have now determined the water accessibility of the M2 transmembrane domain (M2-TM) in virus-envelope-mimetic lipid membranes and its changes with environment Site-specific water-protein magnetization transfer indicates that, in the absence of amantadine, the initial spin diffusion rate mainly depends on the radial position of the residues from the pore pore-lining residues along the helix have similarly high water accessibilities compared to lipid-facing residues Upon drug binding, the spin diffusion rates become much slower for Gly(34) in the middle of the helix than for the N-terminal residues, indicating that amantadine is bound to the pore lumen between Gly(34) and Val(27) Water-protein spin diffusion buildup curves indicate that spin diffusion is the fastest in the low-pH open state, slower in the high-pH closed state, and the slowest in the high-pH amantadine-bound state Simulations of the buildup curves using a 3D lattice model yielded quantitative values of the water-accessible surface area and its changes by pH and drug binding. These data provide direct experimental evidence of the pH-induced change of the pore size and the drug-induced dehydration of the pore This study demonstrates the capability of H-1 spin diffusion NMR for elucidating water interactions with ion channels, water pores, and proton pumps and for probing membrane protein conformational changes that involve significant changes of water-accessible surface areas