There have been speculations that the regulatory (R) subunit of the cAMP-dependent protein kinase (PKA) may have other functions. A recent study has shown that the catalytic (C) subunit of PKA may be regulated in a cAMP- and R subunit-independent manner. However, evidence linking a function to the R subunit apart from inhibiting the C subunit has been elusive. In this report, interaction cloning experiments showed that the RIalpha subunit association with the cytochrome c oxidase subunit Vb (CoxVb) is cAMP-sensitive. Interaction was detected with a GST-RIalpha fusion protein as well as by coimmunoprecipitation. Transient treatment with cAMP-elevating agents inhibited cytochrome c oxidase in Chinese hamster ovary (CHO) cells with a concomitant decrease in cytochrome c levels in the mitochondria and an increase in its release into the cytosol. Furthermore, mutant cells harboring a defective RIalpha show increased cytochrome c oxidase activity and also constitutively lower levels of cytochrome c in comparison to either the wild-type cells or the C subunit mutant. These results suggest a novel mechanism of cAMP signaling through the interaction of RIalpha with CoxVb thereby regulating cytochrome c oxidase activity as well as the cytochrome c levels.