The total creatine pool size (creatine (Cr) + phosphocreatine (PCr) = [Cr_total]) is crucial for optimal energy utilization in skeletal muscle, especially at the onset of exercise and during intense contractions. The [Cr_total] likely is controlled long-term by 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 [Cr_total] was reduced by ~85% (1% b-guanidinoproprionic acid (-GPA)), or their muscle [Cr_total] was repleted (1% Cr after -GPA depletion). Cr uptake was assessed by skeletal muscle ¹⁴C-Cr accumulation to Cr and PCr using the 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 [Cr_total] to ~15% of normal increased Cr uptake in the soleus (21%) and red gastrocnemius (RG; 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 [Cr_total] even though CrT band density was increased 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 [Cr_total] in the high-oxidative muscle types, but not in the WG. This implies that factors controlling Cr uptake are different among the fiber types. These observations may help explain the influence of initial muscle [Cr_total], time dependency and variations in muscle [Cr_total] accumulation during Cr supplementation.