Shallow-living marine invertebrates use free amino acids as cellular osmolytes, while most teleosts use almost no organic osmolytes. Recently we found unusual osmolyte compositions in deep-sea animals. Trimethylamine N-oxide (TMAO) increases with depth in muscles of some teleosts, skates, and crustaceans (up to 300 mmol/kg at 2900 m). Other deep-sea animals had high levels of (1). scyllo-inositol in echinoderms, gastropods, and polychaetes, (2). that polyol plus beta-alanine and betaine in octopods, (3). hypotaurine, N-methyltaurine, and unidentified methylamines in vestimentiferans from hydrothermal vents and cold seeps, and (4). a depth-correlated serine-phosphate osmolyte in vesicomyid clams from trench seeps. We hypothesize that some of these solutes counteract effects of hydrostatic pressure. With lactate dehydrogenase, actin, and pyruvate kinase, 250 mM TMAO (but not glycine) protected both ligand binding and protein stability against pressure. To test TMAO in living cells, we grew yeast under pressure. After 1 h at 71 MPa, 3.5 h at 71 MPa, and 17 h at 30 MPa, 150 mM TMAO generally doubled the number of cells that formed colonies. Sulfur-based osmolytes which are not correlated with depth, such as hypotaurine and thiotaurine, are probably involved in sulfide metabolism and detoxification. Thus deep-sea osmolytes may have at least two other roles beyond acting as simple compatible osmotica.