The intron-containing gene encoding human ribosomal protein S3a (hRPS3a) was isolated by utilizing a PCR-based strategy to detect a gene-specific intron which was subsequently used as a probe for cloning of the entire gene. The hRPS3a gene is composed of six exons and five introns spanning 5013 bp. As described for other hRP-encoding genes, the promoter lacks a canonical TATA sequence and a defined CAAT box. Primer extension experiments, as well as cell-free transcription, revealed that a cytosine functions as the major transcription start point in a polypyrimidine region, but a guanosine at position -1 was also able to initiate transcription. Hybridization analysis of chromosomal DNA from a panel of human-rodent somatic cell hybrids revealed that hRPS3a is encoded by a single locus in the human genome, present on chromosome 4.