The total creatine pool size [Crtotal; creatine (Cr) + phosphocreatine (PCr)] is crucial for optimal energy utilization in skeletal muscle, especially at the onset of exercise and during intense contractions. The Crtotallikely is controlled by long-term modulation of Cr uptake via the sodium-dependent Cr transporter (CrT). To test this hypothesis, adult male Sprague-Dawley rats were fed 1% Cr, their muscle Crtotalwas reduced by ∼85% [1% β-guanidinoproprionic acid (β-GPA)], or their muscle Crtotalwas repleted (1% Cr after β-GPA depletion). Cr uptake was assessed by skeletal muscle14C-Cr accumulation to Cr and PCr by using hindlimb perfusion, and CrT protein content was assessed by Western blot. Cr uptake rate decreased with dietary Cr supplementation in the white gastrocnemius (WG; 45%) only. Depletion of muscle Crtotalto ∼15% of normal increased Cr uptake in the soleus (21%) and red gastrocnemius (22%), corresponding to 70–150% increases in muscle CrT content. In contrast, the inherently lower Cr uptake rate in the WG was unchanged with depletion of muscle Crtotaleven though CrT band density was increased by 230%. Thus there was no direct relationship between apparent muscle CrT abundance and Cr uptake rates. However, Cr uptake rates scaled inversely with decreases in muscle Crtotalin the high-oxidative muscle types but not in the WG. This implies that factors controlling Cr uptake are different among fiber types. These observations may help explain the influence of initial muscle Crtotal, time dependency, and variations in muscle Crtotalaccumulation during Cr supplementation.