Glucokinase catalyzes phosphoryl group transfer from ATP to glucose to form glucose-6-phosphate in the first step of cellular metabolism. While the location of the ATP-binding site of glucokinase was proposed recently, limited information exists on its conformation or the key amino acids involved in substrate binding. Affinity labeling with phenylglyoxal is used to probe possible Arg residues involved in ATP binding. Electrospray ionization mass spectrometry indicates that reaction of purified glucokinase with phenylglyoxal results in as many as six or seven sites of modification, suggesting nonspecific modification. However, preincubation of glucokinase with glucose followed by reaction with phenylglyoxal reveals only two sites of modification. Glucokinase activity assays show that enzyme preincubated with glucose possesses residual activity corresponding to the fraction of unmodified enzyme observed by mass spectrometry, strongly suggesting that glucokinase preincubated with glucose is specifically labeled and inactivated upon modification by phenylglyoxal. The data support the existing conformational model of glucokinase.