Multidomain transcriptional activators and antiterminators that include PTS regulatory domains (PRDs) were subjected to sequence analyses. All of these transcriptional regulators exhibit one or more N-terminal nucleic acid binding site(s) and two PRD regions. Additionally, we show that the activators contain C-terminal PTS IIB and IIA domains with fully conserved phosphorylation sites (cysteine and histidine, respectively). One activator, LevR has a different domain order than all other activators with a truncated IIA domain preceding (rather than following) the IIB domain, and it has a C-terminal PRD, rather than two adjacent PRDs. Our analyses suggest that the activators and antiterminators arose early, and that domain shuffling either within or between proteins has occurred rarely. The results allow us to propose an evolutionary pathway for the appearance of these transcription factors and to suggest functional significance for these domains and specific residues within them.