Knowledge about the molecular structure of PKA isoforms is substantial. In contrast, the dynamics of PKA isoform activity in living primary cells has not been investigated in detail. Using a High Content Screening microscopy approach, we identified the RIIβ subunit of PKA-II to be predominantly expressed in a subgroup of sensory neurons. The RIIβ-positive subgroup included most neurons expressing nociceptive markers (TRPV1, NaV1.8, CGRP, IB4) and responded to pain eliciting capsaicin with calcium influx. Isoform-specific PKA reporters showed in sensory neuron-derived F11 cells that the inflammatory mediator PGE2 specifically activated PKA-II but not PKA-I. Accordingly, pain sensitizing inflammatory mediators and activators of PKA increased the phosphorylation of RII subunits (pRII) in subgroups of primary sensory neurons. Detailed analyses revealed basal pRII to be regulated by the phosphatase PP2A. Increase of pRII was followed by phosphorylation of CREB in a PKA-dependent manner. Thus, we propose RII phosphorylation to represent an isoform-specific readout for endogenous PKA-II activity in vivo, suggest RIIβ as a novel nociceptive subgroup marker, and extend the current model of PKA-II activation by introducing a PP2A-dependent basal state.