A human creatine transporter (hCRT-BS2M) cDNA clone was isolated from a human brainstem/spinal cord using a PCR and phage plaque hybridization based technique. This clone included an open reading frame of 1,905 base pairs(bp) within a 2,283bp cDNA. Northern blot hybridization detected the expression of corresponding mRNAs most prominently in the skeletal muscle, heart and kidney. Peptide sequence analysis of the hCRT-BS2M protein product revealed 12 putative transmembrane domains. The predicted protein sequence further demonstrates that the hCRT-BS2M has highly conserved amino acid identity with the other members of the sodium dependent plasma membrane transporter family. Transient expression of the hCRT-BS2M in COS-7 cells demonstrates sodium dependent [14C]creatine uptake with a KM value of 14.9 +/- 3.0 microM (n = 5) that is attenuated by creatine and selective structural analogues of creatine.