We isolated cDNA coding a new human riboflavin transporter (hRFT)3, which exhibits 86.7 and 44.1% amino acid identity with hRFT1 and hRFT2, respectively. It was predicted to have 10 putative membrane-spanning domains. The functional characteristics of hRFT3 were examined and compared with those of its isoforms, hRFT1 and hRFT2. Real-time PCR revealed that hRFT3 mRNA was strongly expressed in the brain and salivary gland. hRFT1 mRNA was strongly expressed in the placenta and small intestine, whereas hRFT2 mRNA was most abundantly expressed in the testis and strongly in the small intestine and prostate. hRFT-mediated uptake of [3H]riboflavin was evaluated using human embryonic kidney 293 cells transiently transfected with the cDNA coding each hRFT. The apparent Michaelis-Menten constants of hRFT1, hRFT2, and hRFT3 for riboflavin were 1.38, 0.98, and 0.33 micromol/L, respectively. The hRFT-mediated [3H]riboflavin uptake was independent of extracellular Na+ and Cl(-). Specific uptake of [3H]riboflavin by hRFT2, but not hRFT1 and hRFT3, decreased as extracellular pH was changed from 5.4 to 8.4. The substrate specificities of the hRFT family were similar. hRFT-mediated uptake of [3H]riboflavin was inhibited by some riboflavin analogs, but not D-ribose, organic ions, or other vitamins. The newly isolated hRFT3 may play an important role in brain riboflavin homeostasis. Its amino acid sequence and functional characteristics are similar to those of hRFT1, but not hRFT2.