The rates of phosphodiesterase-promoted hydrolysis of cGMP and cAMP have been measured in intact neuroblastoma N1E-115 cells by determining rates of 18O incorporation from 18O-water into the alpha-phosphoryls of guanine and adenine nucleotides. The basal rate of guanine nucleotide alpha-phosphoryl labeling ranged from 180 to 244 pmol X mg protein-1 X min-1. Sodium nitroprusside (SNP) caused a sustained 3.4-fold increase in this 18O-labeling rate in conjunction with 28- and 50-fold increases in cellular cGMP concentration at 3 and 6 min, respectively. This 18O-labeling rate (795 pmol X mg protein-1 X min-1) corresponded with the sum of the low (1.7 microM) and high (34 microM) Km phosphodiesterase activities assayable in cell lysates which exhibited a combined maximum velocity of 808 pmol X mg protein-1 X min-1 to which the high Km species contributed 84%. This information and the characteristics of the profile of 18O-labeled molecular species indicate that cGMP metabolism was restricted to a very discrete cellular compartment(s) of approximately 12% of the cell volume. Carbachol (1 mM) produced a transient increase (6-fold) in cellular cGMP concentration and a transient increase (90%) in the rate of 18O labeling of alpha-GTP during the first minute of treatment which translates into 30 additional cellular pools of cGMP hydrolyzed in this period. IBMX (1 mM) produced a relatively rapid increase in cellular cGMP (3- to 5-fold) and cAMP (2-fold) concentrations and a delayed inhibition of 18O labeling of guanine and adenine nucleotide alpha-phosphoryls without further elevation of cyclic nucleotide levels. These results indicate that besides inhibiting cyclic nucleotide hydrolysis, IBMX also imparts a time-dependent inhibitory influence on the generation of cyclic nucleotides. The data obtained show that measurement of 18O labeling of guanine and adenine nucleotide alpha-phosphoryls combined with measurements of cyclic nucleotide steady state levels provides a means to assess the rates of cyclic nucleotide synthesis and hydrolysis within intact cells and to identify the site(s) of action of agents that alter cellular cyclic nucleotide metabolism.