Adenosine 3:5-cyclic monophosphate (cAMP) is a key second messenger in signaling pathways governing many cellular processes. To define the subcellular localization and relative abundance of cAMP, we developed a novel immunochemical approach based on acrolein fixation to visualize cAMP within cells. We describe here the fixation and immobilization of cAMP within cells and the production of specific, high titer polyclonal antibodies that recognize cAMP. Relative levels of cAMP immunofluorescence were quantitated in glial cells (oligodendrocytes, astrocytes, Schwann cells, and glioma cells) that were either untreated or treated with activators of endogenous adenylyl cyclase to raise cAMP levels. In treated cells, cAMP immunofluorescence is strongly localized in the perinuclear cytoplasm.Many signals that regulate the growth, development, and metabolism in cells use cAMP as a second messenger (1, 2). Neurotransmitters, growth factors, and hormones signal to appropriate receptors, causing either the stimulation or inhibition of adenylyl cyclase through intermediate guanyl nucleotide regulatory proteins. Adenylyl cyclase catalyzes the conversion of ATP to cAMP, which acts by binding to protein kinase A (PKA 1 ) and causing the release of activated catalytic subunits, or by binding and activating cAMP-gated ion channels (3-6). Activated PKA regulates the function of pre-existing cellular proteins by selective phosphorylation and also regulates the synthesis of new cellular proteins by phosphorylating and altering the activity of transcription factors (7-9).In the oligodendrocyte, the myelinating cell of the central nervous system, cAMP is thought to play independent roles in the regulation of cell growth and differentiation. Precursor cells, which are mobile and proliferative, exit the cell cycle upon exposure to cAMP analogs (10). In oligodendrocyte-lineage cells that are differentiating, exposure to cAMP analogs or activators of adenylyl cyclase increases the rate of differentiation (11,12). Whereas experimentally induced cAMP can have potent regulatory effects in these processes, the role of cAMP in normal oligodendrocyte development is not well understood. Cyclic AMP has similar effects in the peripheral myelinating glia, Schwann cells (13), and it is thought that axonal contact causes an elevation in cAMP, inducing proliferation and differentiation (14).Many questions remain about how cAMP relays signals, due in part to technical limitations that have prevented determination of the subcellular distribution of cAMP or comparison of cAMP levels between single cells. Homogeneous populations of cells may vary in their cAMP content from cell to cell, and conventional assays of cAMP extracted from homogenates of such a population generate a simple average level for the set (15). These assay procedures also mask any useful information about cell-specific changes in cAMP levels in the heterogeneous environments of tissue or cell culture. Finally, there have been no methods for accurately measuring free cAMP levels within ...