We report the discovery and characterization of a novel nucleolar protein. This protein, referred to as pNO40 based on its molecular weight on SDS-PAGE, was identified through yeast two hybrid interaction screen of a human kidney cDNA library using pinin (pnn) protein as the bait. The deduced amino acids of pNO40 derived from cDNA cloning of diverse species display high conservation; 95% identify between human and mouse and 57.3% identity for human and zebrafish. Several distinct domains are discernable in the ORF of pNO40, including a ribosomal protein S1 RNA binding region, a CCHC type zinc finger, and clusters of basic amino acid representing potential nucleolar targeting signal. Immunostaining of endogenous or transfected pNO40 indicated that it is localized to nucleoli of diverse cultured cells, with some concentration in the granular component of nucleoli. Northern blot analysis demonstrated that pNO40 message is expressed ubiquitously across all tissues examined. Characterization of human and mouse pNO40 gene revealed that mouse gene spans 44 kb in length and contains 8 exons, while that of human is 68 kb in length and displays two isoforms generated by alternative splicing of the 5(')-untranslated region and differential usage of translation start site. Based on sequence features and its subcellular location, we predict that pNO40 is a novel nucleolar protein with function related to ribosome maturation and/or biogenesis.