A triggering effect of insulin on GLUT4 expression in adipocytes is consistently observed in vivo, whereas GLUT1 is roughly unaffected. However, in cultured rat adipocytes, insulin increases GLUT1 but fails to increase GLUT4, suggesting that additional factors are involved in vivo. This prompted us to evaluate the potential role of glucocorticoids as coregulators with insulin of glucose transporter expression using 3T3-F442A adipose cells and primary cultured rat adipocytes. In both systems, insulin increased and dexamethasone decreased GLUT1 messenger RNA (mRNA) and protein, an effect inhibited by the glucocorticoid antagonist RU 38486. When the two hormones were added together, the effect of dexamethasone was dominant in 3T3-F442A cells, but was totally antagonized in rat adipocytes. Moreover, in rat adipocytes, the GLUT1 gene transcription rate (run-on) was identical in the absence or presence of the two hormones. With regard to GLUT4 expression, neither insulin nor dexamethasone alone had any significant effect after 2 days of treatment. In contrast, the combined hormones markedly increased GLUT4 mRNA (+550% in rat adipocytes; +130% in 3T3-F442A cells) and protein (+164% in rat adipocytes; +79% in 3T3-F442A cells) with a 24- to 48-h delay after mRNA induction. Studies of the molecular mechanism(s) showed that exposure of rat adipocytes to dexamethasone plus insulin did not affect GLUT4 mRNA stability, but increased the GLUT4 gene transcription rate 3-fold. Transient transfections of rat adipocytes with the 5'-flanking 2.2-kilobase sequence of the rat GLUT4 gene fused to luciferase demonstrated that promoter activity was unchanged by insulin, increased 50% by dexamethasone, and increased 3-fold in the presence of both. These data show that insulin elicits an increase in GLUT4 gene expression provided glucocorticoids are present. Our results indicate that the synergism between insulin and glucocorticoids on GLUT4 gene transcription is mediated through GLUT4 promoter activation.