Animal studies have shown that supra-physiological creatine monohydrate (Cr-mH) supplementation for 3 months reduced skeletal muscle creatine transporter (CRT) content. The doses of Cr-mH (1-2 g/kg/day) used in these studies were between 5 and 10 times those usually used in human studies, and it is unclear whether a down-regulation of CRT would occur in humans at the recommended doses of 0.1-0.2 g/kg/day. We measured CRT, and citrate synthase (CS) protein content using Western blotting before and after 2 months of Cr-mH supplementation and weight training in young men (N = 11 Cr-mH (0.125 g/kg/ day); N = 8 placebo). CRT and CS were also measured before and after 4 months of Cr-mH supplementation and weight training in elderly (> 65 years) men and women (N = 14 Cr-mH (0.075 g/kg/day); N = 14 placebo). Finally, CRT mRNA was measured using competitive RT-PCR before and after 8-9 days of Cr-mH loading in young men and women (N = 14, CR-mH (mean = 0.18 g/kg/day); N = 13, PL). Total creatine content was significantly elevated after the Cr-mH supplementation period as compared to placebo in each of the studies. Neither Cr-mH supplementation, nor exercise training resulted in measurable alterations in CRT protein content and acute Cr-mH loading did not alter CRT mRNA. There were no gender differences in CRT mRNA or total creatine content in the young subjects and no gender differences in total creatine content or CRT protein content in the elderly subjects. Weight training in young men did not increase CS protein content, however, in the elderly there was a significant increase in CS protein content after exercise training (p < 0.05). These results demonstrated that Cr-mH supplementation during weight training resulted in increases in skeletal muscle total creatine without reductions in CRT protein and acute Cr-mH loading did not decrease CRT mRNA content.