In eukaryothes, the second messenger cAMP regulates many cellular functions by binding to the regulatory subunits of cAMP-dependent protein kinases, and releasing the catalytic subunits. In the mammalian brain all four regulatory isoforms (RIalpha and beta, RIIalpha and beta) are present. Apparently, they are simple inhibitors of the catalytic subunits. It is still unclear why four isoforms are needed, but possibly they can target kinase activity at precise intracellular locations. Therefore, we examined the distribution of the insoluble regulatory isoforms in rat and mouse brains during prenatal (from embryonic day 8) and postnatal development up to senescence (13 months), via immunohistochemistry. RIIalpha labelling is always restricted to the ventricular ependyma. Punctated RIIbeta labelling is observed in the embryo from early stages of development, and is mainly localized in the cortical plate. After birth, punctate RIIbeta labelling is present throughout almost the whole brain, often observed in proximity of neurofilaments. It shows different characteristics and relationships to the other isoforms: for example in the CA1 hippocampal field, RIIbeta is substituted by RIalpha 2 weeks after birth, while in CA2 it persists for life. In other regions, as in the reticular formation, RIIbeta and RIalpha aggregates are found in the same cell, although clearly segregated. The different regulatory isoforms show distinct patterns of distribution that change consistently during development. A careful characterization of second messenger systems may be as useful as the study of neurotransmitters to understand neuronal properties and their modifications during development, so as to relate biochemical to functional properties.