Some bacteria lack sugar permeases of the bacterial phosphotransferase system (PTS) but encode within their genomes phosphoryl transfer proteins of the PTS that probably function in regulation. These proteins include homologues of HPr (PtsH), the ATP-dependent HPr(ser) kinase/phosphatase (PtsK) and the PEP-dependent HPr(his) kinase known as Enzyme I (PtsI). We identify all currently sequenced homologues of these proteins, multiply align their sequences and construct phylogenetic trees in order to derive functional, structural and evolutionary conclusions. We show that no bacterium possesses more than one HPr kinase and that these proteins are probably all orthologous. alpha-Proteobacteria possess truncated HPr kinases which probably serve a unified regulatory function together with other PTS proteins. The Enzymes I are orthologous in all Gram-positive bacteria and some Gram-negative bacteria, but other Gram-negative bacteria exhibit paralogues that fall into 5 functional types. No bacterium with a fully sequenced genome exhibits all of these types. With the exception of the classical Enzymes I, each of these functional types exhibits a distinctive set of accompanying domains, usually with a characteristic domain order. One functional type, the fructose-specific type, includes two phylogenetically different subgroups with different domain orders. The results establish that domain associations occurred early during evolutionary history of the PTS, and that subsequent domain rearrangements occurred rarely. Our findings define the evolutionary histories of these important bacterial proteins and provide guides for functional assignment of PTS-related proteins encoded by genes revealed by genome sequencing.