Negative control in two-component signal transduction results from sensor transmitter phosphatase activity for phospho-receiver dephosphorylation. A hypothetical mechanism for this reaction involves a catalytic residue in the H-box active-site region. However, a complete understanding of transmitter phosphatase regulation is hampered by the abundance of kinase-competent, phosphatase-defective missense substitutions (K+ P phenotype) outside of the active-site region. For the Escherichia coli NarX sensor, a model for the HisKA_3 sequence family, DHp domain K+ P mutants defined two classes. Interaction mutants mapped to the active site-distal base of the DHp helix 1, whereas conformation mutants were affected in the X-box region of helix 2. Thus, different types of perturbations can influence transmitter phosphatase activity indirectly. By comparison, K+ P substitutions in the HisKA sensors EnvZ and NtrB additionally map to a third region, at the active site-proximal top of the DHp helix 1, independently identified as important for DHp-CA domain interaction in this sensor class. Moreover, the NarX transmitter phosphatase activity was independent of nucleotides, in contrast to the activity for many HisKA family sensors. Therefore, distinctions involving both the DHp and the CA domains suggest functional diversity in the regulation of HisKA and HisKA_3 transmitter phosphatase activities.