To elucidate the organization of the human genomic sequences encoding CTP synthetase (CTPS), fragments homologous to the cDNA were isolated from genomic lambda libraries. The fragments cloned were overlapping and cover over 40 kb. Cotransfection of the DNAs into CTPS-deficient, cytidine-requiring CHO mutants can transform them to cytidine-independent growth, indicating that the complete structural gene has been isolated. Direct sequencing and enzymatic amplification of the cloned genomic fragments revealed that the coding sequences are distributed to 19 exons covering about 35 kb. Multiple transcriptional start sites were detected by primer extension in a G + C-rich 5' flanking sequence that is separated from the translational start by an approximately 3-kb intron. A panel of human-rodent somatic cell hybrids and the CTPS cDNA were used to assign the structural gene to the short arm of human chromosome 1. This assignment was further refined through the use of somatic cell hybrids bearing fragments of the short arm of the chromosome, allowing localization to 1p36.11-p31, a region notable for its disruption in many types of tumors.