The mechanism underlying agonist-induced loss of responsiveness to catecholamines and prostaglandins has been investigated in human astrocytoma cells. Pulse-labeling of the cells with [3H] adenine during the time course of exposure to either norepinephrine or prostaglandin E1 (PGE1) demonstrated a reduction of the rate of incorporation of label into cyclic AMP within 5 min after exposure of the cells to either agonist. The loss of responsiveness observed by this technique was essentially agonist-specific during the first 30 min of exposure of the cells to either norepinephrine or PGE1. The rate constant for degradation of cyclic AMP throughout a 60 min exposure to either norepinephrine or PGE1 did not change suggesting that loss of responsiveness is not related to increased phosphodiesterase activity. The results are discussed in terms of a standard theoretical model for the regulation of the steady state level of an intermediate in a reaction sequence in which the rate of synthesis of the intermediate follows zero order kinetics and the rate of degradation follows first order kinetics. The hypothesis is put forth that agonist-induced desensitization is caused by an agonist-specific reduction in the rate of synthesis of cyclic AMP that follows rapidly after the initial stimulation of adenylate cyclase activity.