The acute actions of low and moderate concentrations of catecholamines on cyclic AMP metabolism in SV40-transformed human lung fibroblasts (VA13) were seriously distorted by non-enzymatic destruction of the agonist. Catecholamine destruction, as measured directly with an isotopic method, was slowed by a variety of anti-oxidants and chelating agents. A combination of two anti-oxidants, ascorbate and thiourea, was very effective in protecting isoproterenol in the cell culture system. That is, there was a 10-fold increase in the sensitivity of VA13 to isoproterenol and the duration of action of the catecholamine was greatly prolonged. However, the anti-oxidants did not alter the responses of the cells to prostaglandins. We conclude that any quantitative studies of cyclic AMP responses to catecholamines must address the question of agonist destruction if meaningful results are to be expected. The use of anti-oxidants, especially the combination of ascorbate and thiourea, would appear to be advisable, particularly in situations where the catecholamine concentrations are less than supramaximal.