The cellular mechanism of the action of atrial natriuretic factor (ANF) is thought to involve activation of guanylate cyclase. Increasing evidence shows a direct tubular effect of ANF. Part of the ANF-induced diuresis has been suggested to be due to inhibition of the action of arginine vasopressin (AVP) in the cortical collecting tubule. In this study we investigated the effect of ANF on cyclic nucleotide production in primary cultures of cortical collecting tubule cells immunodissected with a monoclonal antibody. ANF caused a dose-dependent stimulation in cyclic guanosine 3′,5′-monophosphate (cGMP) production; the half-maximal stimulation was observed at 1 nM of ANF. ANF (0.01–100 nM) had no effect on cyclic adenosine 3,5-monophosphate (cAMP) accumulation in cortical collecting tubule cultures. AVP caused a dose-dependent increase in cAMP production, and this effect was not altered by the simultaneous addition of ANF (100 nM). Similarly, ANF-induced cGMP stimulation was not influenced by AVP (10 nM). We conclude that 1) ANF has a direct stimulatory action on cGMP production by cultured cortical collecting tubule cells and 2) any interaction between ANF and AVP is likely to occur at steps distal to cyclic nucleotide formation.