Membrane proteins play an essential role in cellular metabolism, transportation and signal transduction across cell membranes. The scarcity of membrane protein structures has thus far prevented a full understanding of their molecular mechanisms. Preliminary topology studies and residue solvent exposure analysis have the potential to provide valuable information on membrane proteins of unknown structure. Here, a F-19-containing unnatural amino acid (trimethylfluoro-phenylalanine, tfmF) was applied to accomplish site-specific F-19 spin incorporation at different sites in diacylglycerol kinase (DAGK, an Escherichia coil membrane protein) for site-specific solvent exposure analysis. Due to isotope effect on F-19 spins, a standard curve for F-19-tfmF chemical shifts was drawn for varying solvent H2O/D2O ratios. Further site-specific F-19 solvent isotope shift analysis was conducted for DAGK to distinguish residues in water-soluble loops, interfacial areas or hydrophobic membrane regions. This site-specific solvent exposure analysis method could be applied for further topological analysis of other membrane proteins. (C) 2011 Elsevier Inc. All rights reserved.