Cyclic nucleotide metabolism was studied during human monocyte differentiation. Intracellular cAMP increased 17-fold during in vitro differentiation. This increase was due to an increase in the specific activity of adenylate cyclase and a concomitant decrease in the specific activity of cyclic AMP phosphodiesterase. Monocyte adenylate cyclase activity was stimulated by guanine nucleotide, prostaglandin E1, isoproterenol, and epinephrine. Macrophage adenylate cyclase demonstrated less responsiveness to guanine nucleotide and was refractory to stimulation by prostaglandin E1, and to beta-adrenergic receptor agonists. In contrast to cAMP, total intracellular cGMP levels remained constant. Guanylate cyclase was found predominately in the cytosol of monocytes. The specific activity of soluble guanylate cyclase decreased during differentiation, while particulate activity increased more than 40-fold. Cyclic GMP phosphodiesterase activity remained stable during monocyte differentiation. The ratio of cAMP:cGMP increased dramatically from 2:1 in monocytes to 9:1 in macrophages suggesting that cAMP may be an important mediator of differentiation, while cGMP metabolism decreases in the fully differentiated nonproliferating macrophage.