Incubation of human serum with D-[6-3H]glucose resulted in the gradual accumulation of radioactivity in acid-precipitable material. Upon chromatography on Sephadex G-200, radioactivity was found associated with each of the major molecular weight classes of serum protein. Purified human serum albumin was also glucosylated in vitro upon exposure to D-[6-3H]glucose in phosphate-buffered saline. The glucosylated and unmodified albumins were separated by ion exchange chromatography. The physiological significance of these observations in vitro was confirmed by the isolation and quantitation of glucosylated albumin from normal human serum. Glucosylated albumin represents approximately 6 to 15% of total serum albumin in normal adults. The post-translational modification appears to occur by a nonenzymatic process analogous to that responsible for glucosylation of hemoglobin A to hemoglobin AIc, i.e. through Schiff base formation and Amadori rearrangement to a ketoamine derivative.