Nucleotide sugar transporters have long been assumed to be antiporters that exclusively use nucleoside monophosphates as antiport substrates. Here we present evidence indicating that two other types of nucleotide sugar transporters exist that differ in their antiport substrate specificity. Biochemical studies using microsomes derived from Saccharomyces cerevisiae cells expressing either human (h) UGTrel7 or the Drosophila (d) FRC (Fringe connection) transporter revealed that (i) efflux of preloaded UDP-glucuronic acid from the yeast microsomes expressing hUGTrel7 was strongly enhanced by UDP-GlcNAc added in the external medium, but not by UMP or UDP, suggesting that hUGTrel7 may be described as a UDP-sugar/UDP-sugar antiporter, and (ii) addition of UDP-sugars, UDP, or UMP in the external medium stimulated the efflux of preloaded UDP-GlcNAc from the yeast microsomes expressing dFRC to a comparable extent, suggesting that UDP, as well as UMP, may serve as an antiport substrate of dFRC. Antiport of UDP-sugars with these specific substrates was reproduced and definitively confirmed using proteoliposomes reconstituted from solubilized and purified transporters. Possible physiological implications of these observations are discussed.