New evidence indicates that termination of transcription is an important regulatory step, closely related to transcriptional interference and even transcriptional initiation. However, how this occurs is poorly understood. Recently, in vivo analysis of transcriptional termination for the human beta-globin gene revealed a new phenomenon--co-transcriptional cleavage (CoTC). This primary cleavage event within beta-globin pre-messenger RNA, downstream of the poly(A) site, is critical for efficient transcriptional termination by RNA polymerase II. Here we show that the CoTC process in the human beta-globin gene involves an RNA self-cleaving activity. We characterize the autocatalytic core of the CoTC ribozyme and show its functional role in efficient termination in vivo. The identified core CoTC is highly conserved in the 3' flanking regions of other primate beta-globin genes. Functionally, it resembles the 3' processive, self-cleaving ribozymes described for the protein-encoding genes from the myxomycetes Didymium iridis and Physarum polycephalum, indicating evolutionary conservation of this molecular process. We predict that regulated autocatalytic cleavage elements within pre-mRNAs may be a general phenomenon and that functionally it may provide the entry point for exonucleases involved in mRNA maturation, turnover and, in particular, transcriptional termination.