In intact bovine heart mitochondria, cAMP-dependent phosphorylation of 42, 29, 18 and 6.5 kDa proteins was inhibited by carboxyatractyloside. This shows that both mitochondrial cAMP-dependent protein kinase (mtPKA) and its protein substrates are localized at the matrix side of the inner mitochondrial membrane. Proteins of 42, 29, 18, and 6.5 kDa were also bound at the outer surface of mitochondria where they were phosphorylated by the added purified catalytic subunit of PKA. In the cytosol from bovine heart proteins of the above molecular weights were phosphorylated by the cytosolic PKA.