Incubation of Clone 9 cells, a nontransformed rat liver cell line, in the presence of 5 mM azide results in an induction of GLUT1 mRNA which becomes detectable after 3 h of continuous exposure to the agent. In examining the role of on-going protein synthesis in this response, we found that: (i) the induction of GLUT1 mRNA by azide was not inhibited by anisomycin, (ii) exposure to anisomycin alone also resulted in increased GLUT1 mRNA content, and (iii) the increments in GLUT1 mRNA content in the presence of both azide and anisomycin were additive. Following exposure to 30 μM anisomycin, the increase in GLUT1 mRNA content became evident at 1 h, reached a maximum level of 7-fold at 3 h, then slowly decreased but remained elevated at 2-fold control levels at 12 h. Transcription of the GLUT1 gene, estimated by nuclear run-on assay, was stimulated 1.4 ± 0.1 and 1.6 ± 0.2-fold in cells exposed to anisomycin for 1 and 2 h, respectively (p<0.05 for both). Upon inhibition of RNA synthesis by actinomycin D, GLUT1 mRNA content decreased with a half-life of 1.9 ± 0.4 h in control cells, while in contrast, GLUT1 mRNA half-life was 4.6 ± 0.8 h in cells exposed to anisomycin. The induction of GLUT1 mRNA by anisomycin was half-maximal at 3 μM, whereas inhibition of leucine incorporation and stimulation of Stress Activated Protein Kinase (SAPK), measured as c-Jun N-terminal kinase activity, were half-maximal at 0.3 and 0.05 μM anisomycin, respectively. GLUT1 mRNA content was also increased by the protein synthesis inhibitor emetine, and the effect was associated with no stimulation of SAPK activity. Finally, SAPK activity was minimally stimulated in cells exposed to azide. It is concluded that: (1) on-going protein synthesis is not necessary for the induction of GLUT1 mRNA content in response to azide, (2) the induction of GLUT1 mRNA by anisomycin is related to its activity to inhibit protein synthesis, and (3) under basal conditions, a rapidly turning-over putative protein exerts a negative regulatory effect on GLUT1 mRNA expression.