A human clone corresponding to the gene encoding anion exchanger isoform 3 (AE3) (approved gene symbol SLC2C) has been isolated and partially sequenced. Oligonucleotide primers based on this sequence were used in a polymerase chain reaction to specifically amplify a segment of the human gene from a panel of human-rodent somatic cell hybrids, allowing the assignment of AE3 to chromosome 2. To map AE3 more precisely to a cytogenetic band on chromosome 2, the AE3 cosmid was used as a probe in fluorescence in situ hybridization to human metaphase chromosomes. Fractional length measurements were made, and AE3 mapped at high resolution to the cytogenetic band 2q36. A polymorphic dinucleotide (GT/CA)n repeat marker was developed from sequences in the AE3 cosmid and typed on a subset of the CEPH families. Multipoint linkage analysis placed the AE3 gene between D2S128 and D2S126 on a genetic map of chromosome 2, corroborating the chromosomal localization of AE3 obtained by physical mapping methods.