We have demonstrated that infusion of donor rats with fenoldopam, a dopaminergic compound that maintains renal blood flow without increasing blood pressure, induces HO-1 expression and protects kidneys against cold-storage injury when transplanted. Since fenoldopam has vascular and other cellular effects, whether the protection is mediated through HO-1 remains uncertain. To test this, renal tubular (RPTE) cells were preincubated with fenoldopam (0-80 μg/mL) for 18 hours and subjected to 4°C cold storage for 24 hours. Fenoldopam significantly reduced cold-storage injury (e.g., % LDH release, mean ± SE, 53.4 ± 1.3 cold control vs. 16.2 ± 0.5 and 2.2 ± 0.2 for 10 and 80 μg/mL fenoldopam respectively (n = 3; p < .001). Similarly, inclusion of fenoldopam in UW solution during cold storage also protected the cells against cold-storage injury. The fenoldopam's protections of cells on both of these occasions were associated with a significant increase in HO-1 protein. To test whether HO-1 induction is directly responsible for fenoldopam's cytoprotection in RPTE cells, we employed siRNA oligonucleotides specifically directed against HO-1 mRNA to silence the HO-1 gene. Using real-time RT PCR, we first confirmed that fenoldopam caused a two-fold induction of HO-1 mRNA. In a separate experiment in which RPTE cells were stored in the cold, siRNA dose dependently knocked down the fenoldopam-induced HO-1 mRNA by 70%. The controls, oligofectamine, the vehicle for siRNA, or siRNA alone did not affect HO-1. To determine the effect of HO-1 on fenoldopam's protection, we repeated the experiment in the presence of HO-1 siRNA and measured LDH release. As before, fenoldopam significantly suppressed cold-induced LDH release in these cells. However, the addition of HO-1 siRNA completely abrogated the protective effect of fenoldopam (% LDH release: cold control 50.2 ± 0.4 vs. cold plus fenoldopam 10.1 ± 0.5, vs. cold plus fenoldopam 40 μg/mL plus HO-1 siRNA 52.2 ± 0.3; p < .001) demonstrating a central role for HO-1 in fenoldopam's cytoprotection. Use of fenoldopam may prove to be useful in reducing the cold-storage injury of transplanted organs.