1. Abnormal renal sodium handling is considered a major contributing factor in hypertension associated with chronic ouabain treatment. However, the molecular mechanisms involved in abnormal renal sodium handling have not been elucidated. Therefore, we investigated whether chronic ouabain treatment perturbs dopamine D(1) receptor function. 2. The expression and phosphorylation levels of the D(1) receptor in cells of the human proximal tubule cell line (HK-2) were determined using western blot analysis and reverse transcription polymerase chain reaction. The activity of the renal sodium/potassium pump (Na(+)/K(+)-ATPase) was measured using a colourimetric assay, and cyclic adenosine monophosphate accumulation was determined by performing a radioimmunoassay. 3. We showed that chronic ouabain treatment decreased the protein and mRNA expression levels of the D(1) receptor and increased the basal phosphorylation of the D(1) receptor in HK-2 cells. We also showed that in the presence of ouabain, HK-2 cells did not reveal the cyclic adenosine monophosphate accumulation and Na(+)/K(+)-ATPase inhibition induced by the D(1) receptor agonist fenoldopam. 4. We hypothesize that the ouabain-induced decrease in renal D(1) receptor function is responsible for the increase in renal sodium reabsorption, which eventually leads to ouabain-induced hypertension.