Feeding rats beta-guanidinopropionic acid (beta-GPA), a creatine analogue, results in depletion of creatine and phosphocreatine and induces increases in mitochondrial oxidative enzymes and hexokinase in skeletal muscle. Comparisons of different muscle types and studies of the adaptation to exercise suggest that 1) the levels of the insulin-responsive glucose transporter (GLUT-4), mitochondrial oxidative enzymes, and hexokinase may be coregulated and 2) GLUT-4 content can determine maximal glucose transport activity in muscle. To further evaluate these possibilities, we examined the effects of feeding rats 1% beta-GPA in their diet for 6 wk on muscle GLUT-4 expression and glucose transport activity. beta-GPA feeding induced 40-50% increases in cytochrome c concentration, citrate synthase activity, and hexokinase activity in plantaris muscle. GLUT-4 protein concentration was increased approximately 50% in plantaris and epitrochlearis muscles, while GLUT-4 mRNA was increased approximately 40% in plantaris muscles of beta-GPA-fed rats. Glucose transport activity maximally stimulated by insulin was increased in parallel with GLUT-4 protein concentration in the epitrochlearis. These results provide evidence that chronic creatine depletion increases GLUT-4 expression by pretranslational mechanisms. They support the hypothesis that the levels of mitochondrial enzymes, hexokinase, and GLUT-4 protein are coregulated in striated muscles. They also support the concept that the GLUT-4 content of a muscle determines its maximal glucose transport activity when the signaling pathways for glucose transport activation are intact.