Intron 1 of the coxl gene of yeast mitochondrial DNA (al1) is a group HA intron that encodes a maturase function required for its splicing in vivo. It is shown here to self-splice in vitro under some reaction conditions reported earlier to yield efficient self-splicing of group IIB introns of yeast mtDNA that do not encode maturase functions. Unlike the group IIB introns, al1 is inactive in 10 mM Mg2+ (including spermidine) and requires much higher levels of Mg2+ and added salts (1M NH4CI or KCI or 2M (NH4)2SO4) for ready detection of splicing activity. In KCI-stimulated reactions, splicing occurs with little normal branch formation; a post-splicing reaction of linear excised intron RNA that forms shorter lariat RNAs with branches at cryptic sites was evident in those samples. At low levels of added NH4CI or KCI, the precursor RNA carries out the first reaction step but appears blocked in the splicing step. AM RNA is most reactive at 37–42°C, as compared with 45°C for the group IIB Introns; and it lacks the KCl-or NH4CI-dependent spliced-exon reopening reaction that is evident for the self-splicing group IIB introns of yeast mitochondria. Like the group IIB intron al5γ, the domain 4 of ah can be largely deleted in cis, without blocking splicing; also, frans-splicing of half molecules interrupted in domain 4 occurs. This is the first report of a maturase-encoding intron of either group I or group II that self-splices in vitro.