Introns constitute most of the length of typical pre-mRNAs in vertebrate cells. Thus, the turnover rate of introns may significantly influence the availability of ribonucleotides and splicing factors for further rounds of transcription and RNA splicing, respectively. Given the importance of intron turnover, it is surprising that there have been no reports on the half-life of introns from higher eukaryotic cells. Here, we determined the stability of IVS1Cβ1, the first intron from the constant region of the mouse T-cell receptor-β (TCR-β) gene. Using a tetracycline (tet)-regulated promoter, we demonstrate that spliced IVS1Cβ1 and its pre-mRNA had half-lives of 6.0 ± 1.4 min and 3.7 ± 1.0 min, respectively. We also examined the half-lives of these transcripts by using actinomycin D (Act.D). Act.D significantly stabilized IVS1Cβ1 and its pre-mRNA, suggesting that Act.D not only blocks transcription but exerts rapid and direct posttranscriptional effects in the nucleus. We observed that in vivo spliced IVS1Cβ1 accumulated predominantly as lariat molecules that use a consensus branchpoint nucleotide. The accumulation of IVS1Cβ1 as a lariat did not result from an intrinsic inability to be debranched, as it could be debranched in vitro, albeit somewhat less efficiently than an adenovirus intron. Subcellular-fractionation and sucrose-gradient analyses showed that most spliced IVS1Cβ1 lariats cofractionated with pre-mRNA, but not always with mRNA in the nucleus. Some IVS1Cβ1 also appeared to be selectively exported to the cytoplasm, whereas TCR-β pre-mRNA remained in the nucleus. This study constitutes the first detailed analysis of the stability and fate of a spliced nuclear intron in vivo.