We investigated the specificity of CAAX box-related isoprenylation of rod photoreceptor cGMP phosphodiesterase (PDE) subunits expressed in bacteria and the consequences of this modification on rod disk membrane association. Full- length cDNA sequences of the α and β subunits of mouse PDE, inserted into bacterial pET expression vectors, were overexpressed as fusion proteins containing 28 (bMP-α) and 26 (bMP-β) additional amino acid residues at their N termini. Both fusion proteins were overexpressed and stored in inclusion bodies. Purified bMP-α and bMP-β were recognized by bovine PDE- specific polyclonal antibodies, but did not associate with depleted rod disk membranes and were catalytically inactive. Using bovine brain or retina extracts as sources of protein prenyltransferases and tritiated farnesyl- or geranylgeranylpyrophosphate as donors, bMP-α (CAAX sequence CCIQ) was exclusively farnesylated, and bMP-β (CAAX sequence CCIL) was exclusively geranylgeranylated. After isoprenylation, bMP-α and bMP-β each associated with rod photoreceptor outer segment disk membranes under isotonic, but not under hypotonic, conditions. The results indicate that isoprenylated bMP-α and bMP-β each interact independently with membranes and that isoprenylation is the key modification that facilitates membrane association.