We have previously demonstrated that, by elevating intracellular adenosine 3':5'-cyclic monophosphate (cAMP) levels by inhibition of cAMP phosphodiesterase with Ro 20-1724 or by forskolin stimulation of adenylcyclase, the growth of neoplastically transformed 10T1/2 fibroblasts could be inhibited when these cells were in contact with growth-inhibited nontransformed 10T1/2 cells (J. S. Bertram and M. B. Faletto, Cancer Res., 45: 1946-1952, 1985) and furthermore that the extent of this growth inhibition correlated strongly with the degree of junctional communication between the two cell types (P.P. Mehta et al., Cell, 44: 187-196, 1986). To determine if these treatments enhance the degree of growth control of the nontransformed 10T1/2 cells, cultures were exposed to varying concentrations of Ro 20-1724 and/or forskolin. Drug treatment caused no significant effects on growth rate or cell spreading when cells were treated during logarithmic growth phase; however, major reductions of up to 70% in confluent saturation density and concomitant increases in cell spreading occurred in cultures making extensive cell/cell contacts. Decreases in saturation density correlated strongly with induced elevations of both intra- and extracellular cAMP concentrations. These effects could not be duplicated by the addition of exogenous cAMP agonists 8-bromo-cAMP and/or dibutyryl-cAMP. Two-dimensional electrophoresis of phosphate-labeled proteins revealed that forskolin treatment induced a quantitatively and qualitatively different phosphorylation profile than did 8-bromo-cAMP. Both basal and drug-induced intracellular cAMP levels fell as cells progressed from logarithmic to confluent growth state, implying that cells become sensitized to cAMP by the attainment of extensive cell/cell contacts. It is suggested that the drug-induced elevations of endogenously synthesized cAMP are accentuating a physiological role of cAMP on the postconfluent growth arrest of murine fibroblasts. The requirements for cell/cell contact and the known increased junctional communication induced by cAMP furthermore suggest that cAMP is enhancing the junctional transfer of a growth-inhibiting regulatory molecule. A likely candidate is cAMP itself.