Chinese hamster ribosomal protein S14 cDNA was used to recognize homologous human cDNA and genomic clones. Human and Chinese hamster S14 protein sequences deduced from the cDNAs are identical. Two overlapping human genomic S14 DNA clones were isolated from a Charon 28 placental DNA library. A fragment of single-copy DNA derived from an intron region of one clone was mapped to the functional RPS14 locus on human chromosome 5q by using a panel of human X Chinese hamster hybrid cell DNAs. The human S14 gene consists of five exons and four introns spanning 5.9 kilobase pairs of DNA. Polyadenylated S14 transcripts purified from HeLa cell cytoplasma display heterogeneous 5' ends that map within noncoding RPS14 exon 1. This precludes assignment of a unique 5' boundary of RPS14 transcripts with respect to the cloned human genomic DNA. Apparently HeLa cells either initiate transcription at multiple sites within RPS14 exon 1, or capped 5' oligonucleotides are removed from most S14 mRNAs posttranscription. In contrast to the few murine ribosomal protein and several other mammalian housekeeping genes whose structures are known, human RPS14 contains a TATA sequence (TATACTT) upstream from exon 1. Three related short sequence motifs, also observed in murine and yeast ribosomal protein genes, occur in this region of the RPS14 gene. RPS14 introns 3 and 4 both contain Alu sequences. Interestingly, the Alu sequence in intron 3 is located slightly downstream from a chromosome 5 deletion breakpoint in one human X hamster hybrid clone analyzed.