The four previously characterized genes (Surf1 to 4) of the mouse Surfeit locus do not share any sequence homology, and the transcription of each gene alternates with respect to its neighbor(s). Adjacent Surfeit genes are separated by very small distances, and two of the genes overlap at their 3' ends. In this work we have further defined the Surfeit gene cluster by the isolation of Surf5, a fifth gene of the locus, and determination of its relationship to the other Surfeit genes. Surft5 does not share any sequence homology with the four cloned Surfeit genes. The transcription of Surf5 is divergent with respect to its neighbor the Surf3 gene, and the 5' ends of Surf5 and Surf3 are separated by only 159 bp, suggesting the presence of a second bidirectional promoter in the locus. The 3' end of Surf5 maps only 68 bp away from the processed 3' end of a pseudogene. The human and partial chicken Surf5 coding regions show greater than 95% identity, and a Caenorhabditis elegans homologue shows 38% identity and 56% similarity with the mouse Surf5 amino acid sequence. The 3.5-kb transcript of Surf5 encodes a small hydrophilic protein of 140 amino acid residues, which differs from the ribosomal protein L7a encoded by the Surf3 gene or the integral membrane protein encoded by the Surf4 gene. Subcellular fractionation located the Surf5 protein to the soluble fraction of the cytoplasm. The surfeit locus appears to represent a novel type of gene cluster in which the genes are unrelated by sequence or function; however, their organization may play a role in their gene expression.