The PII proteins compose a superfamily of signal transducers with fundamental roles in the nitrogen metabolism of prokaryotic organisms. They act at different cellular targets, such as ammonia transporters, enzymes, and transcriptional factors. These proteins are small, highly conserved, and well distributed among prokaryotes. The current PII classification is based on sequence similarity and genetic linkage. Our work reviewed this classification through an extensive analysis of PII homologues deposited in GenBank. We also investigated evolutionary aspects of this ancient protein superfamily and revised its PROSITE signatures. A new group of PII proteins is described in this work. These PII homologues have a peculiar genetic context, as they are associated with metal transporters and do not contain the canonical PROSITE signatures of PII. Our analysis reveals that horizontal gene transfer could have played an important role in PII evolution. Thus, new insights into PII evolution, a new PII group, and more comprehensive PROSITE signatures are proposed.