Using a cloned cDNA copy of T-cell growth factor (TCGF) mRNA from the Jurkat leukemic T-cell line, we have isolated three overlapping TCGF genomic clones from a human DNA library. The entire TCGF gene is contained within two adjacent EcoRI fragments spanning about 8 kilobases. The complete nucleic acid sequence was determined. The gene is divided into four exons. The 5' untranslated region and the first 49 amino acids of the protein, 20 of which constitute a signal polypeptide and are not present in the secreted protein, are encoded by the first exon. Exons 2 and 3, separated from each other by a long intervening sequence, contain coding information for the next 20 and 48 amino acids, respectively. The remaining 36 amino acids and the 3' untranslated region are contained in the fourth exon. A promoter sequence T-A-T-A-A-A is present 77 base pairs (bp) upstream from the translation initiation site, and a CAT homology region occurs 104 bp upstream from the initiation site. A putative site for initiation of mRNA transcription was identified 53 bp 5' of the translation initiation codon. The organization of the gene was shown by Southern blot analysis to be identical in normal peripheral blood lymphocytes and in a variety of malignant lymphoid cell types. Restriction analysis of these cellular DNAs produced results exactly as predicted by the map for the cloned genomic TCGF, indicating that there is only a single copy of the human TCGF gene.