The accumulation of endogenous adenosine 3':5'-cyclic phosphate (cAMP) and of [(14)C]cAMP, derived from nucleotides labeled by prior incubation of cells with [(14)C]adenine, has been investigated in four glial cell lines from rats. The following results are similar to data reported for brain slices: (i) rat glial cells contain a system that readily incorporates [(14)C]adenine into nucleotides that serve as precursors for cAMP. (ii) norepinephrine stimulates accumulation of both [(14)C]cAMP and endogenous cAMP, and (iii) the phosphodiesterase inhibitors, papaverine and isobutylmethylxanthine, are effective in enhancing the stimulatory effect of norepinephrine. In contrast to results reported for brain slices, histamine, veratridine, and adenosine, the purine either alone or with a biogenic amine, do not cause an enhanced accumulation of cAMP in rat glial cells, and repetitive accumulations of cAMP can be elicited during a series of restimulations of the cells with norepinephrine. The magnitude of the accumulation of cAMP elicited by the catecholamine decreases markedly by the fifth restimulation in the absence of, but only slightly in the presence of, papaverine. In glial cells, a large portion of the [(14)C]adenine is incorporated into intracellular compounds that do not serve as precursors of [(14)C]cAMP.