When 3T3-L1 fibroblasts differentiate to adipocytes, the specific activity of pyruvate carboxylase (PC) increases about 25-fold in parallel with its intracellular protein concentration. The increase in PC protein concentration is accompanied by a 9-10-fold increase in the relative abundance of 4.2 kb PC mRNA measured by Northern-blot analysis using a cDNA probe encoding a segment of the PC gene of 3T3-L1 adipocytes. The effects of cyclic AMP (cAMP) alone and together with insulin on levels of cellular protein, PC activity, PC protein and on the relative abundance of PC mRNA were examined in mature 3T3-L1 adipocytes. Adipocytes exposed to cAMP for 24 h exhibited a 25% decrease in cellular protein and marked decreases in enzyme activity (88%) and PC mRNA abundance (98%) compared with untreated adipocyte controls. After 48 h of exposure to cAMP, PC activity and PC mRNA diminished to levels approaching their detection limits. When exposed to medium containing cAMP plus insulin, adipocyte enzyme activity and PC mRNA declined more slowly during the first 24 h exposure (about 20% decrease) but after 48 h fell to values comparable with those of adipocytes exposed to cAMP alone. Despite these decreases in enzyme activity, the PC protein content of adipocytes treated with cAMP alone or cAMP plus insulin are nearly identical with that of control adipocytes. The inactivation of PC in cAMP-treated adipocytes does not involve loss of the prosthetic group from the holoenzyme. Cross-linking experiments suggest that the spatial arrangement of protomers in inactive PC may differ from that in the active tetrameric enzyme. Data presented suggest that, in addition to inducing inactivation, cAMP may also regulate adipocyte PC by decreasing transcription of the PC gene and/or enhancing the rate of degradation of PC mRNA.