A 75-kb region encompassing the human annexin III (ANX3) gene on chromosome 4q21 was characterized from directly amplified genomic DNA and from six genomic clones in phage lambda (lambda ANX3-1 to lambda ANX3-6). The gene was mapped with restriction enzymes BamHI, EcoRI, HindIII, SacI, and XbaI, and primers were developed for nine sequence-tagged sites throughout the gene. The transcribed region spans 58 kb and contains 12 introns ranging from 0.3 to 19.1 kb and 13 exons ranging from 53 to 374 bases. Northern blots showed a single mRNA species of approximately 1.7 kb in all tissues examined. Three intragenic polymorphisms were identified: a tandem repeat of the sequence TAAA with at least six alleles, a two-allele SalI polymorphism detectable by polymerase chain reaction, and a two-allele BglII polymorphism.