Inhibition of proximal tubular phosphate (Pi) reabsorption involves, as far as we know, brush border membrane retrieval of the type IIa Na/Pi-cotransporter. The aim of the present study was to analyze whether intracellular cGMP-mediated regulation of Pi reabsorption also involves retrieval of the type IIa Na/Pi-cotransporter, as previously shown for cAMP. Atrial natriuretic peptide (ANP) and nitric oxide (NO) were used to stimulate guanylate cyclase. In vivo perfusion of mice kidneys with either ANP or NO donors resulted in a downregulation of type IIa Na/Pi-cotransporters on the brush border membranes of proximal tubules. These effects were mimicked by activation of protein kinase G with 8Br-cGMP. In in-vitro-perfused mice proximal tubules, ANP was effective when added either to the apical or basolateral perfusate, suggesting the presence of receptors on both membrane sites. The effects of ANP and NO were blocked by the protein kinase G inhibitor LY 83553. Parallel experiments in OK cells, a renal proximal tubule model, provided similar information. Our findings document that cGMP-mediated regulation (ANP and NO) of type IIa Na/Pi-cotransporters also takes place via internalization of the transporter protein.