HIF Prolyl-4-hydroxylase domain-containing proteins (PHDs) promote the degradation of Hypoxia Inducible Factor-1 (HIF-1). Given that HIF-1 is highly expressed in the renal medulla and that HIF-1-targeted genes such as nitric oxide synthase, cyclooxygenase and heme oxygenase are important in the regulation of renal medullary function, we hypothesized that PHD regulates HIF-1 levels in the renal medulla and thereby participates in the control of renal sodium excretion. The present study demonstrated that all 3 isoforms of PHD including PHD1, PHD2 and PHD3 were expressed in the kidneys using Real-Time RT-PCR, Western blot and immunohistochemical analyses, and that PHD2 was the most abundant isoform. Regionally, all PHDs exhibited much higher levels in the renal medulla than in the renal cortex. Increase in renal medullary tissue PO₂ by furosemide, significantly decreased PHD levels in renal medulla, while hypoxia caused significant increases in mRNA levels of PHDs in cultured renal medullary interstitial cells, indicating the oxygen regulation of PHDs. Functionally, a PHD inhibitor, L-mimosine (50 mg/kg/day, i.p. for 2 weeks), substantially upregulated the expressions of HIF-1 in the kidneys, especially in the renal medulla, and remarkably enhanced the natriuretic response to renal perfusion pressure by >80% in Sprague-Dawley rats. Renal medullary transfection of HIF-1 decoy oligodeoxynucleotides to inhibit HIF transcriptional activity attenuated L-mimosine-induced enhancement in pressure natriuresis, which confirmed that HIF-1-mediated the effect of L-mimosine. These results indicate that highly expressed PHDs in the renal medulla importantly contribute to the control of renal sodium excretion through regulation of HIF-1 and its targeted genes.