A cDNA clone encoding human nucleolar phosphoprotein B23, numatrin, was isolated from a library prepared with mRNA from human U cells. The complete nucleotide sequence was determined; it revealed a single open reading frame of 294 amino acids that included four in-frame AUG codons as potential sites of translation initiation. Comparison of the human B23 nucleotide sequence with the rat and mouse B23 sequences revealed 91% homology (hum:rat, and hum:mus) in the coding region; the predicted B23 proteins displayed 94% amino acid identity. Northern gel blot analysis revealed a single B23 mRNA species of approximately 1.5 kb. The level of B23 mRNA in U cells was not detectably altered by treatment with either alpha or gamma interferon. Southern gel blot analysis revealed polymorphism within the human B23 gene structure, and suggested the presence of multiple B23 genes and/or extensive splicing of B23 RNA transcripts.