The cyclic AMP binding proteins present in mouse, rat, bovine, and sheep brains were compared. Extracts were isotopically labeled with 8-azido-cyclic [32P]AMP, a photoaffinity analog specific for cyclic AMP binding sites, and then subjected to two-dimensional gel electrophoresis. The resulting autoradiographic patterns were generally similar, but showed consistent species variations. Proteins identified by their size and phosphorylatability as regulatory subunits of Type II protein kinase isozymes were present in all species, but with slight variations in pI. A series of charge variants identified as regulatory subunits of Type I kinase isozymes on the basis of their size was also ubiquitously present, as were several smaller proteins postulated to be proteolytic fragments derived from the regulatory subunits. The major species difference was a series of labeled proteins found only in rodent brains and not in the brains of any ruminant, or in other rodent tissues. These proteins had a molecular weight of 54,000 and a pI range of 5.89--6.26, and could not be endogenously phosphorylated. The identities of these proteins and their relationship to the protein kinase regulatory subunits are unknown.