Hormone responsiveness is mediated by signal transduction mechanisms involving second messengers, such as cAMP and Ca2+, which regulate reversible changes in the phosphorylation state of proteins. During senescence individuals frequently exhibit a diminished responsiveness to hormones. We examined changes in enzymes involved in protein phosphorylation reactions that might account for this decreased adaptiveness in old mice, and observed the following post-maturational changes: (1) cAMP-dependent protein kinase (Pk-A) specific activity decreased in spleen cytosol and in the particulate fractions of lung, spleen and liver of 24-month-old mice as compared to 2-month-old mice. Splenic cytosolic Pk-A activity decreased by 18 months of age, while particulate activity decreased by 6 months; (2) The amount of 8-N3-[32P]cAMP, a photoaffinity analog of cAMP, incorporated into Pk-A regulatory (R)-subunits from spleen and liver particulate fractions decreased, while photolabeling of R-subunit degradative products with this analog in heart and spleen cytosol increased. (3) Age-dependent increases in membrane-associated protease activities were found in all organs, along with a decrease in cytosolic lung calpain activity. These proteolytic changes may account for the enhanced R-subunit degradation and decreased Pk-A activities observed during senescence. (4) Age-dependent alterations in Ca2+/phospholipid-dependent protein kinase (Pk-C) are organ specific: lung, liver, brain, and heart demonstrate no change in Pk-C activity, while spleen exhibits decreased activity. We hypothesize that these age-dependent alterations in kinase and proteolytic activities may be in part responsible for changes in cellular response to hormonal stimulation, differentiation signals, and antigen responsiveness during senescence.