Newly synthesized small nuclear RNA (snRNA) species U1 and U2 are easily identified in cytoplasmic fractions prepared by standard aqueous cell fractionation. However, because the mature stable snRNA species leak from isolated nuclei during cell fractionation, the possibility exists that these newly synthesized species also leak from the nucleus. To overcome the problems of nuclear leakage, mouse L929 cells were fractionated by cell enucleation. Enucleation extrudes the nuclei from cytochalasin-treated cells and produces cytoplasts that, by several criteria, are a bona fide cytoplasmic fraction uncontaminated by nuclear material. All six of the major snRNAs are present in the cytoplasts (c-snRNAs) shortly after synthesis. The species are identified by immunoprecipitation with specific antisera against the ribonucleoproteins and by Northern blotting and hybrid selection using cloned probes. This confirms and extends similar studies that used non-aqueous cell fractionation and manual dissection to overcome nuclear leakage. Kinetic studies demonstrate that the c-snRNAs return to the interphase nucleus after approximately 20 minutes in the cytoplasm. The U2 precursor U2' is processed to mature-sized U2 in the cytoplast fractions before returning to the nucleus. The c-snRNAs occur in ribonucleoprotein particles with similar antigenicity to the mature nuclear particles within six minutes of transcription. This suggests that in mammalian cells, important steps in the assembly of these ribonucleoproteins occur in the cytoplasm.