Adenosine 3',5'-cyclic monophosphate (CAMP) content of neurons is determined not only by the rate of synthesis but also by the rate of hydrolysis by cyclic nucleotide phosphodiesterases. Multiple forms of cyclic nucleotide phosphodiesterase exist in brain and other tissues, and these may be regulated by various hormones and neuromodulators. The present study examines this regulation in a cloned line of neuroblastoma cells (N18TG2). A biphasic Lineweaver-Burk plot of CAMP hydrolysis revealed two K, s approximating 5 and 25 pM. Lineweaver-Burk plots of cGMP hydrolysis were linear over a range of 1 pM to 1 mM and exhibited a K, of 37 pM. Neither CAMP nor cGMP competed for hydrolysis of the alternative cyclic nucleotide. No evidence for an allosteric activation of CAMP phosphodiesterase by cGMP was found. Calcium regulation of phosphodiesterase was not found in spite of preparation of the cell extract with several protease inhibitors, and addition of exogenous calmodulin. No effect of calmodulin antagonists (calmidazolium, W7, or trifluoperazine) was observed in vitro or in situ. Growth of the cells in the presence of 200 nM 3,5,3'-triiodothyronine (T3) resulted in an increased hydrolysis of CAMP but not of cGMP. This increase was attributed to an increase in V,,, with no change in either high or low K, . This response was blocked by cycloheximide, suggesting that the thyroid hormone effect requires protein synthesis. The thyroid hormone response in neuroblastoma cells is compared with the results of other studies of thyroid hormone effects on phosphodiesterase in other tissues in vivo.