The macronuclear rRNA genes of Tetrahymena thermophila contain a 0.4 kb intervening sequence. In this paper we present evidence that the excision of the intervening sequence from pre-rRNA occurs in vitro in isolated T. thermophila nuclei. The transcription-processing system includes alpha-amanitin to inhibit non-rRNA synthesis and aurintricarboxylic acid to inhibit endogenous nucleases. A discrete 0.4 kb RNA comprises up to 6% of the RNA synthesized in this system. Southern hybridization with restriction fragments of the rDNA (rRNA genes) shows that the 0.4 kb RNA contains the intervening sequence. The size of the 0.4 kb RNA, 410-425 nucleotides, is the size predicted if the entire intervening sequence were excised as a single linear molecule. The 0.4 kb RNA accumulates post-transcriptionally and appears to be stable in vitro. T. pigmentosa strain 6UM, whose rDNA has an intervening sequence of the same size and location as that of T. thermophila, also produces a 0.4 kb RNA in vitro; T. pigmentosa strain 8ALP, with not rDNA intervening sequence, produces no such RNA. The measurement of the accumulation of the excised intervening sequence is a convenient assay for the pre-rRNA splicing activity and a means for characterizing some of the details of the reaction. The resistance of the splicing acticity to concentrations of aurintricarboxylic acid that inhibit other endogenous nucleases should be useful in assaying the splicing enzyme during purification.