Recombinant DNA probes, produced by the molecular cloning of immunoglobulin K light chain mRNA, have been used to analyze heterogeneous nuclear RNA for presumptive precursors to cytoplasmic K light chain mRNA. Three discrete classes of nuclear RNA containing K mRNA sequences were detected after pulse-labeling of immunoglobulin-producing P3 myeloma cells. Two of these were substantially larger than K mRNA (approximately 10 and 4 times larger); the third was similar in size to K mRNA. Beginning with the largest, the sequential appearance of these three classes of nuclear RNA preceded the first a pearance of newly synthesized K light chain mRNA in the cytoplasm. The results presented here suggest that immunoglobulin K light chain mRNA is generated by the stepwise cleavage and processing of a large nuclear RNA transcript. Resolution of the events in the biogenesis of mRNA is essential to an understanding of the regulation of gene expression in eukaryotic cells. It is now well established that most mRNAs in eukaryotic cells are derived from nuclear RNA through post-transcriptional modifications including poly(A) addition (1-3), methylation, and the addition of 5'-terminal "caps" of the general structure m7G(5')pppN'-m-N"(m)p (4-6). Considerable evidence also suggests that the mRNA in eukaryotic cells is generated by the post-transcriptional cleavage of larger nuclear RNA (2,3,6). Compelling results now indicate that viral mRNA from the DNA tumor viruses simian virus 40 (7) and adenovirus (8-11) and -the cellular mRNA for globin (12)(13)(14)(15) are processed from substantially larger nuclear RNA. However, all mRNA in eukaryotic cells may not be processed from large nuclear RNA. RNA molecules larger than the respective cytoplasmic mRNA were not detected for ovalbumin mRNA (16) and murine RNA tumor virus RNA (17). However, short-lived nuclear RNAs, particularly those only marginally larger than their respective cytoplasmic mRNAs, may not be detected under the labeling and hybridization conditions generally used. In the case of silk fibroin mRNA, pulse-labeling at reduced temperature (presumably lowering the rate of RNA cleavage) was necessary to reveal a short-lived presumptive precursor only slightly larger than the silk fibroin mRNA (18)(19)(20) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. K light chain mRNA* by procedures previously established for the preparation of recombinant DNA clones from rabbit globin mRNA (22). Using cloned immunoglobulin light chain recombinant plasmid DNA, we have detected discrete classes of rapidly labeled nuclear RNA containing K mRNA sequences which, after stringent denaturation (25, 26), remain substantially larger than the K light chain mRNA in immunoglobulin-producing mouse myeloma cells. Kinetic labeling studies indicate that immunoglobulin K light chain mRNA appears to be processed from a large nuc...