Using PCR cloning we isolated the first intron of the human ribosomal protein S6 gene (hRPS6). By screening the human HeLa cell cDNA library in lambda ZAPII vector (Stratagene, La Jolla, CA), we identified and sequenced a partially spliced pre mRNA copy of hRPS6. The complete hRPS6 gene was isolated from a lambda DASH library with an intron-specific probe. The gene and flanking regions were sequenced, and the mRNA 5' end was mapped by primer extension experiments. The hRPS6 gene has 6 exons and 5 introns and is 3.6 kb long. Using intron-specific primers in PCR and a panel of human-hamster cell lines we localized the hRPS6 gene in human chromosome 9.