In higher eukaryotes the large number of introns present in most genes implies that the pre-mRNA processing machinery should be efficient and accurate. Although this could be achieved at the level of each intron, an attractive alternative would be that interactions between introns improve the performance of this machinery. In this study we tested this hypothesis by comparing the processing of transcripts of the tumor necrosis factor beta gene, which differ only by their number of introns. We took advantage of the ordered splicing of the three introns present in this gene to design constructs that should generate, as primary transcripts, molecules that are normally produced by splicing. We established that the apparent splicing rate of intron 3 is increased 2.5- and 3.5-fold by the presence of one or two other introns on the primary transcript, respectively. Similarly, the apparent splicing rate of intron 2 is increased by the presence of intron 1. As these effects involve the splice sites of the upstream intron, these observations support the existence of cooperative interactions between introns during pre-mRNA processing.