Many transport proteins of bacteria and eukaryotes are thought to possess a common structural motif of 12 transmembrane-spanning alpha-helical segments. In this report we use statistical methods to establish that five families or clusters of these facilitators comprise a single superfamily. The five clusters include: (1) drug-resistance proteins, (2) sugar facilitators, (3) facilitators for Krebs cycle intermediates, (4) phosphate ester-phosphate antiporters and (5) a distinct group of oligosaccharide-H+ symporters. Over 50 transporters of bacteria, lower eukaryotes, plants and animals, and one putative bacterial transcriptional regulatory protein are members of this superfamily, which we term the 'major facilitator superfamily' (MFS).