The amino acyl sequences of eight permeases (enzymes II and enzyme II‐III pairs) of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) have been analyzed. All systems show similar sizes, and six of these systems exhibit the same molecular weight ±2%. Several exhibit sequence homology. Characteristic NH2‐terminal and COOH‐terminal sequences were found. The NH2‐terminal leader sequences are believed to function in targeting of the permeases to the membrane, whereas the characteristic COOH‐terminal sequences are postulated to mediate interaction with the energy‐coupling protein phospho HPr. One of the systems, the one specific for mannose, exhibits distinctive characteristics. A pair of probable phosphorylation sites was detected in each of the five most similar systems, those specific for β‐glucosides, sucrose, glucose,N‐acetylglucosamine, and mannitol. One of the two equivalent phosphorylation sites (proposed phosphorylation site 1) was located approximately 80 residues from the COOH terminus of each system. The other site (proposed phosphorylation site 2) was located approximately 440 residues from the COOH termini of the glucose andN‐acetylglucosamine systems, approximately 320 residues from the COOH termini of the β‐glucoside and sucrose systems, and 381 residues from the COOH terminus of the mannitol system. Intragenic rearrangement during evolutionary history may account for the different positions of phosphorylation sites 2 in the different PTS permeases. More extensive intragenic rearrangements may have given rise to entirely different positions of phosphorylation in the glucitol, mannose, and lactose systems. A single, internal amphipathic α‐helix with characteristic features was found in each of seven of the eight enzymes II. The lactose‐specific enzyme III ofStaphylococcus aureuswas unique in possessing a COOH‐terminal amphipathic a‐helix rich in basic amino acyl residues. Possible functions for these amphipathic segments are discussed.— Saier, M. H., Jr.; Yamada, M.; Erni, B.; Suda, K.; Lengeler, J.; Ebner, R.; Argos, P.; Rak, B.; Schnetz, K.; Lee, C. A.; Stewart, G. C.; Breidt, F., Jr.; Waygood, E. B.; Peri, K. G.; Doolittle, R. F. Sugar permeases of the bacterial phosphoenolpyruvate‐dependent phosphotransferase system: sequence comparisons.FASEB J.2: 199‐208; 1988.