1. Rat glioma C6 cells continuously cultured undergo a degeneration in their catecholamine-responsive adenylate cyclase system. Beta-adrenergic receptor number decreases and the cells do not accumulate cyclic AMP when exposed to Beta-agonists. Activity of catecholamine-stimulated adenylate cyclase is lower but not NaF-, GTP-, or cholera toxin-stimulated activities. 2. Prolonged exposure of C6 cells or human fibroblasts to Beta-agonists results in desensitization. The cells no longer respond to the agent but still respond to cholera toxin and, for the fibroblasts, to prostaglandin E1. In contrast, fibroblasts exposed to PGE1 become desensitized not only to that agent but also to catecholamines and cholera toxin. Thus, Beta-agonists and PGE1 have different mechanisms of desensitization. 3. Macrophages exposed to cycloheximide lose their sensitivity to cholera toxin in parallel to inhibition of protein synthesis. The cells retain their toxin receptors and sensitivity to Beta-agonists. In addition, adenylate cyclase can still be activated by the A1 subunit of the toxin in cell membranes. As cycloheximide treatment blocks degradation of iodotoxin, the presence of a membrane component involved in transmembrane translocation of the toxin (or A1) is indicated by these results.