We have isolated, characterized, and sequenced the gene coding for the 72-kDa human high affinity IgG FcR, hFc gamma RI. It consists of six exons and spans 9.4 kilobase pairs. The leader sequence is encoded by two exons, the second of which is 21 base pairs long and contains the predicted site of peptidase cleavage. The third, fourth, and fifth exons each encode homologous Ig-like extracellular domains. The hydrophobic transmembrane region and a highly charged cytoplasmic tail are encoded by a single final exon. The sequence of the 5'-flanking region was determined. Two major transcription initiation sites were identified by RNase protection. The first, more downstream site was confirmed by primer extension studies; canonical CAAT and TATA boxes are located in appropriate positions upstream from this site. The second transcription initiation site was utilized only in RNA from cells incubated with gamma-interferon. This site initiates transcription upstream from the first major site. These data are consistent with the finding of two species of mRNA for hFc gamma RI in myeloid cells that are upregulated when cultured with gamma-interferon. Southern analysis of genomic DNA confirms the restriction map generated from the cloned DNA. One additional HindIII fragment was observed in genomic DNA from 13 randomly selected individuals that was not present in the phage clone used to characterize the gene. This observation suggests the existence of a second hFc gamma RI gene which lacks one of the two internal HindIII sites rather than a restriction fragment length polymorphism.