Streptozotocin (STZ), an analog of N-acetylglucosamine (GlcNAc), is a specific toxin for the pancreatic β cell. We found that treatment of rats with STZ results in an early β-cell-specific increase in the level of intracellular protein modification by O-linked GlcNAc (O-GlcNAc). Using a model O-GlcNAc peptide based on the transcription factor Sp1, we show that treatment of cultured cells with STZ during peptide biosynthesis results in hyperglycosylation of the peptide as a result of the ability of STZ to specifically inhibit the activity of O-GlcNAc-selective N-acetyl-β-D- glucosaminidase. Although this inhibitory activity of STZ probably can occur in all cells, we found, using in situ hybridization, that β cells express very high levels of the mRNA encoding the enzyme responsible for cytoplasmic protein O-glycosylation, O-GlcNAc transferase (OGT). These findings suggest that the pancreatic β cell is particularly sensitive to the toxicity of STZ because it expresses such high levels of OGT. When STZ blocks O-GlcNAc removal from intracellular proteins, the cell with the most rapid on-rate for O-GlcNAc, the β cell, will experience the most rapid accumulation of this protein modification. Because we also show that the on-rate of O-GlcNAc is substrate driven in several cell types, we speculate that the β cell, with its high level of OGT, may also respond to elevations of blood sugar with increased protein modification by O-GlcNAc. Thus, this proposed mechanism of STZ toxicity on the β cell may result from an exaggeration of a heretofore unrecognized physiological response to glucose mediated through the high level of OGT in these cells.