The cAMP receptor protein and cAMP-dependent protein kinase activity in rabbit peritoneal neutrophils have been identified. The cAMP receptor protein in either the plasma membrane or cytosol fractions, identified by photoaffinity labeling with 8-N3-[32P]cAMP, has an apparent molecular weight of 54,000. The cytosol and membrane receptor proteins have apparent dissociation constants for 8-N3-[32P]cAMP of 0.20 microM and 0.06 microM, respectively. The molecular weight and dissociation constant for 8-N3-[32P]cAMP of this cAMP receptor protein are similar to what has been known for RII, the regulatory subunit of the type II cAMP-dependent protein kinase. Unlike the human neutrophils, no evidence of RI activity was detected. cAMP-dependent protein kinase activity was identified by using histone as a substrate. Subcellular fractionation studies showed that the cAMP receptor protein and the cAMP-dependent protein kinase activity are most enriched in the cytosol fraction.