Components I and II (CI&II) in frog rod outer segments (ROS) are prominent cAMP-dependent protein kinase (PK-A) substrates. Their phosphorylation level is high in the dark, and illumination causes dephosphorylation. In order to understand their physiological role in phototransduction, biochemical characterization of CI&II phosphorylation was performed. Fractionation of phosphorylated ROS proteins showed that CI&II in the soluble fraction were highly phosphorylated by endogenous PK-A, whereas those in the membrane-associated protein fractions were not. The latter proteins could be phosphorylated by purified catalytic subunit of PK-A (PK-Acat) while the former proteins were not, suggesting that membrane-bound CI&II are normally much less phosphorylated. Treatments that dissociate the alpha subunit (alpha t) of transducin (Gt) from beta gamma subunits (beta gamma t) and thus produce excess free subunits of Gt in the soluble fraction caused inhibition of CI&II phosphorylation in the soluble fraction and enhancement of CI&II phosphorylation in the peripheral membrane fractions containing less Gt. Unphosphorylated CI&II tightly associated with the washed ROS membranes could be extracted after phosphorylation by PK-Acat. Phosphorylation also caused elution of beta gamma t from the membrane under the same conditions. Cross-linking by the maleimidobenzoyl-N-hydroxysuccinimide ester of the peripheral membrane fraction produced a distinct phosphorylated 50 kDa product with concurrent disappearance of the beta subunit of transducin (beta t) and phosphorylated CI&II. This phosphorylated cross-linked product was not recognized by a monoclonal anti-alpha t antibody but was recognized by antiserum against beta t, suggesting that the 50 kDa protein is a complex of beta gamma t and CI&II. Amino terminal sequencing of components I and II suggests that they are identical proteins with a unique sequence unrelated to other proteins in protein data bases. Phosphopeptide mapping of phosphorylated CI&II in the soluble fraction yielded two trypsinized phosphopeptides, while that in the peripheral membrane fractions showed only one phosphopeptide. These data suggest that multiple phosphorylation of CI&II alters their cellular localization. We conclude that phosphorylation of CI&II controls their localization in frog ROS and an interaction of CI&II with subunits of Gt regulates their phosphorylation.