Alcohol based cosolvents, such as trifluoroethanol (TFE) have been used for many decades to denature proteins and to stabilize structures in peptides. Nuclear magnetic resonance spectroscopy and site directed mutagenesis have recently made it possible to characterize the effects of TFE and of other alcohols on polypeptide structure and dynamics at high resolution. This review examines such studies, particularly of hen lysozyme and β-lactoglobulin. It presents an overview of what has been learnt about conformational preferences of the polypeptide chain, the interactions that stabilize structures and the nature of the denatured states. The effect of TFE on transition states and on the pathways of protein folding and unfolding are also reviewed. Despite considerable progress there is as yet no single mechanism that accounts for all of the effects TFE and related cosolvents have on polypeptide conformation. However, a number of critical questions are beginning to be answered. Studies with alcohols such as TFE, and ‘cosolvent engineering’ in general, have become valuable tools for probing biomolecular structure, function and dynamics.1. COSOLVENTS: OLD HAT? 2982. HOW DOES TFE WORK? 2992.1 Effect on hydrogen bonding 3002.2 Effect on non-polar sidechains 3012.3 Effect on solvent structure 3023. EFFECTS OF TFE ON (UN-)FOLDING TRANSITIONS 3033.1 Pretransition 3033.2 Transition 3043.3 Posttransition 3053.4 Far UV CD spectroscopic detection of structure 3063.5 Effect with temperature 3063.6 Effect with additional denaturants 3064. THERMODYNAMIC PARAMETERS FROM STRUCTURAL TRANSITIONS OF PEPTIDES AND PROTEINS IN TFE 3075. ADVANCES IN NMR SPECTROSCOPY 3105.1 Chemical shifts 3105.2 ³[Jscr ]_HNHαcoupling constants 3115.3 Amide hydrogen exchange 3125.4 Nuclear Overhauser Effects (NOEs) 3126. α-HELIX – EVERYWHERE? 3136.1 Intrinsic helix propensity equals helix content? 3136.2 A helix propensity scale for the amino acids in TFE 3146.3 Capping motifs and stop signals 3156.4 Limits and population of helices as seen by CD and NMR 3167. TURNS 3178. β-HAIRPINS AND SHEETS 3179. ‘CLUSTERS’ OF SIDECHAINS 32010. THE TFE DENATURED STATE OF β-LACTOGLOBULIN 32111. THE TFE DENATURED STATE OF HEN LYSOZYME 32412. TERTIARY STRUCTURE, DISULPHIDES, DYNAMICS AND COMPACTNESS 32713. PROSPECTS FOR STRUCTURE CALCULATION 32814. EFFECT OF TFE ON QUATERNARY STRUCTURE 32915. EFFECT ON TFE ON UN- AND REFOLDING KINETICS 33016. OTHER USES 33616.1 Mimicking membranes and protein receptors 33616.2 Solubilizing peptides and proteins 33616.3 Cosolvents as helpers for protein folding? 33816.4 Modifying protein dynamics and catalysis 33816.5 Effects on nucleic acids 33916.6 Effects on lipid bilayers and micelles 33916.7 Future applications 33917. CONCLUSIONS: TFE – WHAT IS IT GOOD FOR? 34018. ACKNOWLEDGMENTS 34019. REFERENCES 340