The heat shock response involves activation of heat shock transcription factor 1 (Hsf1) followed by the rapid synthesis of the protective heat shock proteins (Hsps). To determine if the stress experienced during streptozotocin (STZ)-induced diabetes altered the heat shock response, male Sprague-Dawley rats (n = 33; 280–300 g) were assigned to 4 groups: (1) control, (2) diabetic (30 days after 55 mg/kg STZ i.v.), (3) heat stressed (42°C for 15 minutes), and (4) diabetic heat-stressed group (heat stressed 42°C for 15 minutes, 30 days after 55 mg/kg STZ i.v.). The content of Hsp72, Hsp25, and Hsf1 in skeletal muscles, heart, kidney, and liver was assessed by Western blotting, while electrophoretic mobility shift gel analysis was used to assess Hsf activation. Without heat stress, the constitutive expression of Hsp25, Hsp72, and Hsf1 in tissues from diabetic animals and controls was similar. However, 24 hours following heat stress, the heart, kidney, and liver from diabetic animals showed an increased Hsp72 and Hsp25 content compared to the same tissues from heat-stressed nondiabetic animals (P < 0.05). The white gastrocnemius and plantaris muscles from heat-stressed animals (diabetic and nondiabetic) both showed significant and similar elevations in Hsp72 content. Interestingly, while all muscles from nondiabetic animals showed significant (P < 0.05) increase in Hsp25 content after heat stress, no increase in Hsp25 content was detected in muscles from heat-stressed diabetic animals. As expected, Hsf activation was undetectable in all tissues from non–heat-stressed animals but was detectable in tissues from both diabetic and nondiabetic animals following heat stress with the exception of diabetic skeletal muscle, where it was attenuated. Hsf1 content was unaltered in all tissues examined except in the white gastrocnemius muscles from heat-stressed diabetic animals. where it was undetectable. These results suggest that when tissues from STZ-induced diabetic animals are heat stressed, the Hsp/stress response is altered in a tissue-specific manner. This impaired ability to activate the stress response may explain, at least in part, the selective atrophy of certain muscles or muscle fiber types during diabetes.