The Surfeit locus contains the tightest cluster of mammalian genes so far described. The five Surfeit genes (Surf-1 to -5) that have been previously isolated and characterized do not share any DNA or amino acid sequence homology. These Surfeit genes appear to be housekeeping genes, with the Surf-3 gene encoding the 1.7a ribosomal protein and the Surf-4 gene encoding an integral membrane protein most likely associated with the endoplasmic reticulum. In this work, we have isolated the Surf-6 gene, a sixth member of the Surfeit locus. The Surf-6 gene contains four exons spanning a genomic region of 14 kb and specifies a mRNA of 2,571 bases. Surf-6 has features common to housekeeping genes because its transcript is present in every tissue tested, its 5' end is associated with a CpG-rich island, and its promoter does not contain a canonical TATA box. The Surf-6 long open reading frame encodes a novel highly basic polypeptide of 355 amino acids (28% Arg and Lys). By immunofluorescence and immunoblot analyses, the Surf-6 protein has been found to be located in the nucleolus and by immunocytochemical microscopy to be localized predominantly in the nucleolar granular component, a structure that is involved in ribosome maturation. These results indicate that the novel Surf-6 gene is involved in a nucleolar function.