The complete nucleotide sequence of the circular mitochondrial (mt) DNA of the chlorophyte alga Prototheca wickerhamii has been determined (55,328 base-pairs, A+T content 74·2%). The genes identified encode three subunits of the cytochome oxidase, apocytochrome b, nine subunits of the NADH dehydrogenase complex (nad1 to 7, nad4L and nad9), three ATPase subunits (atp6, atp9, atp1 (also referred to as atpA)), three ribosomal RNAs (5 S (rrn5), small subunit (srn) andlarge subunit (lrn) RNA), 26 tRNAs, and 13 ribosomal proteins. A total of five group I introns reside in lrn and cox1, two of which include intronic open reading frames (ORFs). Five free-standing ORFs longer than60 codons are present. Three of these ORFs are counterparts to genes encoding proteins of unknown functionin plant mitochondria (orf25 and orfB of angiosperms and orf244 of liverwort), whereas two of them are unique. Mitochondrial genes are encoded on both DNA strands in a way that suggests the existence of two transcription units, each including approximately one halfof the mitochondrial genome. The two intergenic regions in which transcription is believed to initiate andterminate are about ten times longer than the other intergenic regions (1118 and 1993 nt versus 100 to 150 nt). A total of 29 recurring sequence motifs (30 to 200 nt long) have been found in intergenic regions. Nine different types of motifs are present, most of them arranged as tandem repeats. These motifs may be implicated in transcription, e.g. as signals forinitiation, termination and/or processing. Phylogenetic analysis on the basis of the cox1 gene strongly suggested that P. wickerhamii and plant mitochondrial genomesare monophyletic. The finding of plant-specificmitochondrial genes such as orf25, orf244, orfB and rrn5 in P. wickerhamii mitochondria corroborates thisidea. © 1994 Academic Press, Inc.