Glutamine (Gln) protects gut mucosa against injury and promotes mucosal healing. Because the induction of heat shock proteins (HSP) protects cells under conditions of stress, we determined whether Gln conferred protection against stress in an intestinal epithelial cell line through HSP induction. Gln added to IEC-18 cells induces an increase in HSP70, a concentration-dependent effect also seen with mRNA. Two forms of injury, lethal heat (49 degrees C) and oxidant, were used, and viability was determined by 51Cr release. Gln-treated cells were significantly more resistant to injury. Treatment with 6-diazo-5-oxo-L-norleucine (DON), a nonmetabolizable analog of Gln, induced HSP70 and protected cells from injury, but less than Gln. These findings suggest that the effects of Gln on HSP70 induction and cellular protection are mediated by metabolic and nonmetabolic mechanisms. To determine whether HSP induction was central to the action of Gln and DON, quercetin, which blocks HSP induction, was used. Quercetin blocked HSP70 induction and the protective effect of Gln and DON. We conclude that the protective effects of Gln in intestinal epithelial cells are in part mediated by HSP70 induction.