The binding of ^[3H]cAMP to cytosol and membrane fractions o f the anterior pituitary of sheep exposed to mild electrical stress was investigated. The fractions were incubated with different concentrations (10-200 nM) of [³H]cAMP; the bound ligand was precipitated with ammonium sulphate and isolated on Millipore filters. [³H]cAMP binding was evaluated by Scatchard analysis. In control animals, the dissociation constant (K_D) was 29.13±3.41 nM and 21.2±2.0 nM in membrane and cytosol fractions, respectively. In the membrane fraction, one-day stress decreased K_D to 12.42±1.64 nM, whereas prolongation of stress to three days resulted in a return of K_D to control levels. Stress had no influence on the K_D in cytosol fractions. The maximal binding activity (B_max) of [³H]cAMP was about three-fold higher in the membrane fraction (148.4±12.0 pmol/pituitary) than in the cytosol (45.00±1.25 pmol/pituitary) in unoestrous, untreated ewes. One-day stress exposure resulted in a decrease in B_max of [³H]cAMP in membrane and cytosol fractions. Prolongation of stress to three days caused a return of B_max to near control levels in both fractions. These results indicate that, in the anterior pituitary gland, stress may influence, probably via hormonal stimulations, the activity of protein kinase A through modifying the affinity of its regulatory subunit for cAMP and regulatory subunit contents. The membrane-bound regulatory subunit of protein kinase A is three-fold more abundant than the cytosolic regulatory subunit.