The structure of the gene encoding ZN-16, a transcription factor that binds to the mammalian growth hormone promoter in tandem with Pit-1, was determined in order to elucidate the exon-intron organization of the 16 zinc finger domains of the protein. Southern hybridization of mouse genomic DNA showed fragments with sizes identical to those predicted from mouse ZN-16 cDNA for two different probes covering the 2200 aa coding frame. Mouse genome database sequences also showed no introns in the zn-16 coding regions on chromosome 4. Analysis of human zn-16 by Southern hybridization and genomic database sequence analysis also indicated a single exon for the human protein coding sequences. BLASTP query of available genomic databases with critical zinc finger residues from mouse ZN-16 identified highly similar canine, bovine, and chimpanzee genomic sequences that encode proteins. Phylogenetic analysis of these mammalian proteins resulted in relationships as would be expected in species spanning rodents to humans. All six independent zn-16 sequences show a single exon coding region with no introns, a similarity ruling out the possibility that these genomic sequences are pseudogenes. Thus, mammalian zn-16 has a compact single exon structure encoding a very large protein (2200-3000 aa), the conservation of which may have functional implications such as the importance of posttranscriptional modifications.