This paper combines two techniques-mass spectrometry and protein charge ladders to examine the relationship between the surface charge and hydrophobicity of a representative globular protein (bovine carbonic anhydrase II; BCA II) and its rate of amide hydrogen deuterium (H/D) exchange. Mass spectrometric analysis indicated that the sequential acetylation of surface lysine-epsilon-NH3+ groups-a type of modification that increases the net negative charge and hydrophobicity of the surface of BCA II without affecting its secondary or tertiary structure resulted in a linear decrease in the aggregate rate of amide H/D exchange at pD 7.4, 15 degrees C. According to analysis with MS, the acetylation of each additional lysine generated between 1.4 and 0.9 additional hydrogens that are protected from HID exchange during the 2 h exchange experiment at 15 degrees C, pD 7.4. NMR spectroscopy demonstrated that none of the hydrogen atoms which became protected upon acetylation were located on the side chain of the acetylated lysine residues (i.e., lys-epsilon-NHCOCH3) but were instead located on amide NHCO moieties in the backbone. The decrease in rate of exchange associated with acetylation paralleled a decrease in thermostability: the most slowly exchanging rungs of the charge ladder were the least thermostable (as measured by differential scanning calorimetry). This observation that faster rates of exchange are associated with slower rates of denaturation is contrary to the usual assumptions in protein chemistry. The fact that the rates of H/D exchange were similar for perbutyrated BCA II (e.g., [lys-epsilon-NHCO(CH2)(2)CH3](18)) and peracetylated BCA II (e.g., [lys-e-NHCOCH3](18)) suggests that the electrostatic charge is more important than the hydrophobicity of surface groups in determining the rate of H/D exchange. These electrostatic effects on the kinetics of H/D exchange could complicate (or aid) the interpretation of experiments in which H/D exchange methods are used to probe the structural effects of non-isoelectric perturbations to proteins (i.e., phosphorylation, acetylation, or the binding of the protein to an oligonucleotide or to another charged ligand or protein).