Nucleoline (100 kDa) is the major nucleolar protein in exponentially growing cells that behaves like a nucleolar organizer protein and plays a key role in rDNA transcription and prerRNA processing. We reported the isolation of 5 cDNA clones by probing a cDNA library, constructed in the expression vector lambda gt11, with a polyclonal serum raised against nucleoline. A new immunoassay, using hybrid proteins (beta gal-cDNA encoded protein) was developed to establish that the isolated cDNAs encoded parts of nucleoline. A further confirmation resulted from the sequence comparison between the cDNA encoded peptide and a 42 aa peptide isolated from rat nucleoline (1). The 5 cDNAs overlapped extensively and covered more than 90% of a full length cDNA. By probing a Northern blot with the 100 kDa cDNA, a 2650 nucleotide polyA+ RNA was detected that contained just enough information to code for nucleoline.