Inbred mouse strains display marked differences in avidity for sweet solutions due in part to genetic differences among strains. Using several techniques, we have located a number of regions throughout the genome that influence sweetener acceptance. One prominent locus regulating differences in sweetener preferences among mouse strains is the saccharin preference (Sac) locus on distal chromosome 4. Afferent responses of gustatory nerves to sweeteners also vary as a function of allelic differences in the Sac locus, suggesting that this gene may encode a sweet taste receptor. Using a positional cloning approach, we identified a gene (Tas1r3) encoding the third member of the T1R family of putative taste receptors, T1R3. Introgression by serial back-crossing of a chromosomal fragment containing the Tas1r3 allele from the high sweetener-preferring strain onto the genetic background of the low sweetener-preferring strain rescued its low sweetener-preference phenotype. Tas1r3 has two common haplotypes, one found in mouse strains with elevated sweetener preference and the other in strains relatively indifferent to sweeteners. This study, in conjunction with complimentary recent studies from other laboratories, provides compelling evidence that Tas1r3 is equivalent to the Sac locus and that the T1R3 receptor (when co-expressed with taste receptor T1R2) responds to sweeteners. However, other sweetness receptors may remain to be identified.