Microtubule-associated protein 2 (MAP2) binds, and is a substrate for, type II cAMP-dependent protein kinase. The structural domain in MAP2 that binds the regulatory subunit (RII) of protein kinase II was identified by expressing fragments of a human MAP2 cDNA in E. coli using the pATH11 vector. Fusion proteins were resolved by SDS-PAGE and transferred to nitrocellulose. The filters were probed with purified bovine heart or brain RII, anti-RII monoclonal antibodies, and 125I-labeled protein A. Binding of RII was localized to a 31 amino acid sequence near the N-terminus of the MAP2 molecule. Fusion proteins containing this fragment bound both heart and brain RIIs in a concentration-dependent manner, but bound heart RII with a higher apparent affinity than brain RII. The amino acid sequence of this fragment (DRETAEEVSARIVQVVTAEAVAVLKGEQEKE) is totally conserved between human and mouse MAP2, suggesting an important role for the RII binding site of MAP2 in neuronal function.