Streptozotocin (STZ), an analog of N-acetylglucosamine (GlcNAc), is a specific toxin for the pancreatic beta cell. We found that treatment of rats with STZ results in an early beta-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-beta-D-glucosaminidase. Although this inhibitory activity of STZ probably can occur in all cells, we found, using in situ hybridization, that beta 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 beta 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 beta 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 beta 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 beta cell may result from an exaggeration of a heretofore unrecognized physiological response to glucose mediated through the high level of OGT in these cells.