Three signalling pathways lead to activation of the phosphate (Pho) regulon by phosphorylation of the response-regulator PhoB in Escherichia coil One pathway responds to the extracellular inorganic phosphate (Pi) level and leads to activation by the Pi sensor kinase, PhoR. The other two pathways are Pi independent and are apparent in the absence of PhoR. One Pi-independent pathway responds to the level of an unknown catabolite and leads to activation by the catabolite regulatory sensor kinase, CreC (originally called PhoM); the other Pi-independent pathway responds to acetyl phosphate and leads to activation by a process requiring acetyl phosphate. Here we show that activation of PhoB by acetyl phosphate can require the sensor kinase EnvZ. Accordingly, we propose that the in vivo activation of PhoB by acetyl phosphate (and perhaps other two-component response-regulators as well) probably always requires a certain kinase that can vary depending upon the growth conditions.