Glutamine is considered a non-essential amino acid, however, it becomes conditionally essential during critical illness when consumption exceeds production. Glutamine may modulate the heat shock/stress response, an important adaptive cellular response for survival. Glutamine increases heat-induction of Hsp25 in both epithelial IEC-18 and mesenchymal NIH/3T3 cells, an effect that is neither glucose nor serum dependent. Neither arginine, histidine, proline, leucine, asparagine, or tyrosine acts as physiological substitutes for glutamine for heat induction of Hsp25. The lack of effect of these amino acids was not caused by deficient transport, although some amino acids, including glutamate (a major direct metabolite of glutamine), were transported poorly by IEC-18 cells. Glutamate uptake could be augmented in a concentration- and time-dependent manner by increasing either media concentration and/or duration of exposure. Under these conditions, glutamate promoted heat induction of Hsp25, albeit not as efficiently as glutamine. Further evidence for the role of glutamine conversion to glutamate was obtained with the glutaminase inhibitor, 6-diazo-5-oxo-L-norleucine (DON) which inhibited the effect of glutamine on heat-induced Hsp25. DON inhibited phosphate-dependent glutaminase by 75% after 3 hours, decreasing cell glutamate. Increased glutamine/glutamate conversion to glutathione was not involved as the glutathione synthesis inhibitor, buthionine sulfoximine, did not block glutamine's effect on heat-induction of Hsp25. A large drop in ATP levels did not appear to account for the diminished Hsp25 induction during glutamine deficiency. In summary, glutamine is an important amino acid and its requirement for heat-induced Hsp25 supports a role for glutamine supplementation to optimize cellular responses to pathophysiological stress.