DiSSU1 is an optional group I twintron present in the nuclear extrachromosomal ribosomal DNA of the myxomycete Didymium iridis. DiSSU1 appears to be complex both in structure and function. At the RNA level it has a twin-ribozyme organization composed of two group I ribozymes with different functions, separated by an open reading frame. Here, we show that DiSSU1 is mobile when haploid intron-containing and intron-less amoebae are mated. The mobility process is fast, being completed in 5-10 nuclear cycles after mating in the developing zygote and plasmodia. Analyses of progeny from genetic crosses confirm intron mobility. DiSSU1 is the first example of a mobile group I twintron. The intron-encoded protein was expressed in Escherichia coli and found to be an endonuclease, I-DirI, that cleaves an intron-less ribosomal DNA allele at the intron-insertion site, and is probably involved in intron homing. The endonuclease I-DirI seems to be a rare example of a protein that is expressed from a ribozyme-processed RNA polymerase I transcript in vivo.