Mutations in the kinase domain of the T-cell receptor-associated protein tyrosine kinase ZAP-70 have been shown to result in a novel form of severe combined immunodeficiency syndrome (SCID) in humans in which peripheral CD8* T cells are absent (Science 264:1596-1599). Those results demonstrated that ZAP-70 is required for normal T-cell signaling and function. Furthermore, ZAP-70 is critical to normal T-cell development and intrathymic selection of a major subset of peripheral T cells in humans. In order to study the role of ZAP-70 expression in T-cell ontogeny as well as to identify ZAP-70 mutations in patients with SCID who lack ZAP-70 mRNA, we cloned and sequenced the human ZAP-70 gene. A human placental cosmid library was screened with a 2.2-kb human ZAP-70 cDNA. Five positive clones were identified and subcloned into plasmids for fine mapping and sequence analysis. One clone contained most of the 3′ portion of the gene, but none of the clones contained the ZAP-70 promoter or proximal 5′ gene sequences. In order to obtain the missing part of the gene, the library was rescreened using a unique 300-bp 5′ cDNA PCR fragment as a probe. Seven additional clones were identified and subcloned. Detailed mapping analysis revealed that the human ZAP-70 gene spans at least 40-kb and consists of more than eight exons. Most of the exons are located in the 5′ portion of the gene and correspond to the known protein structure, which includes two src-homology (SH)2 domains. The kinase domain of the ZAP-70 protein is encoded by DNA sequences within the 3′ region of the gene, which is separated from 5′ DNA sequences encoding the two SH2 domains by large introns. Characterization of the human ZAP-70 gene should be useful to the understanding of ZAP-70 regulation in developing T cells as well as in the identification of mutations in some patients with ZAP-70 deficiency.