Abstract We report the isolation and characterization of genomic DNA clones encoding Xenopus ribosomal protein (rp) S6. A human rp S6 cDNA was used to screen a genomic DNA library, and this led to the isolation of a genomic clone encompassing the complete rp S6 gene locus. DNA sequencing and primer extension analysis indicate that Xenopus rp S6 is structurally analogous to the mammalian rp S6 genes, and its transcription starts at two sites within the same polypyrimidine tract of 10 bases. A series of deletions of the 5′ region of the Xenopus rp S6 gene were fused to the chloramphenicol acetyltransferasereporter gene and transfected into COS-1 cells. The results suggest that the regulatory regions of the Xenopus rp S6 gene are clearly distinct from those earlier reported for the human rp S6 gene. Northern blot analysis of stage-specific embryonic RNA demonstrated an uniform rp S6 transcription during embryogenesis. Southern blot and PCR analyses indicate that the Xenopus rp S6 gene is pseudotetraploid in the Xenopus genome.