A 547-bp group-I intron belonging to subgroup IA1 was found near the 3' end of the large subunit ribosomal RNA-encoding gene (LSUrRNA) in the mitochondrial (mt) DNA of the cellular slime mold Dictyostelium discoideum. This intron was inserted in a highly conserved stretch within the sequence that encodes the peptidyl transferase center domain V in the corresponding region of the Escherichia coli LSUrRNA. Interestingly, the insertion site of the intron is the same as that of the So.LSU.2 intron of the green alga, Scenedesmus obliquus, mt DNA and the Pw.LSU.2 intron of the colorless alga, Prototheca wickerhamii, mt DNA. The intron could self-splice in vitro at a concentration higher than 20 mM MgCl2. Polymerase chain reaction analysis showed the possible existence of an intron similar to that of D. discoideum LSUrRNA in another cellular slime mold, Polysphondylium pallidum (CK-8), but not in D. mucoroides (Dm7 and Dm11).