Pleiotrophin (PTN), midkine (MK), and retinoic acid-induced heparin-binding (RI-HB) protein are members of a recently discovered family of developmentally regulated cytokines. We report here the cloning, sequencing, chromosomal localization, and structural organization of the genomic version of the human PTN gene and its comparison to the mouse MK gene. The PTN gene was found to be arranged in five exons and four introns, in a fashion similar to that of the mouse MK gene. Exon 1, as for MK, does not appear to encode amino acid sequence. As in the case of the MK gene, exon 2 encodes the hydrophobic leader sequence of PTN, which constitutes the beginning of gene translation. The signal peptide cleavage site of both genes lies toward the 3' end of exon 2. Exons 3 and 4 of PTN were most closely related to exons 3 and 4 of the MK gene; in particular, six of the ten cysteine residues were coded for in exon 3 and the remaining 4 in exon 4. The intron-exon splice junctions of both genes occurred through the same residues. The two genes were found to be less closely related in the fifth exon which encodes the highly basic C-terminal domains, the translation termination codon, and the polyadenylation signal of both cDNAs. We also report approximately 2000 bp of the 5' untranslated sequence of the PTN gene and the site of initiation of transcription in human placenta. PTN was localized to human chromosome 7q33-34 by fluorescence in situ hybridization. These data confirm the existence of a new gene family of developmentally regulated cytokines.