Glucokinase (GK, EC 2.7.1.2), a member of the enzyme family of hexokinases, has been shown to be linked to maturity-onset diabetes of the young type II (MODY-2). Although nucleotide and amino acid sequence information are available for the human varieties, they are not known for the variety from Bacillus stearothermophilus, which is often used in protein binding studies. Here, a combination of electrospray Fourier transform mass spectrometry (FTMS) and infrared multiphoton dissociation (IRMPD) is used to obtain accurate molecular weight and preliminary amino acid sequence information for the protein. Electrospray FTMS provides evidence of a solution phase dimer. In addition, dithiothreitol reduction shows no shift in high-resolution isotopic distributions, indicating a probable absence of disulfide bonds in the protein. The partial sequence information obtained from IRMPD could be the basis for creating a DNA probe for the protein.