Four human genomic DNA clones for 2', 3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) were isolated by screening a human genomic library with human CNP cDNA clones as probes. Restriction mapping and sequence analysis revealed that the human CNP gene is about 8.5 kb long and composed of four exons interrupted by three introns. There are two transcription start points and in human brain, two forms of CNP mRNA are produced from a single gene by alternative splicing, similar to mouse. A homology search of the 5'-flanking regions of exon 0 and exon 1 in the human CNP gene indicated the presence of oligodendroglia-specific elements and myelin basic protein transcription element (MBTE) motif, in addition to TATA-box-like sequences. Spot blot hybridization of flow-sorted human chromosomes with the 3'-noncoding region of the human CNP cDNA showed the localization of CNP to chromosome 17.