Acute renal failure (ARF) has a high morbidity and mortality. In animal ARF models, effective treatments must be administered before or shortly after the insult, limiting their clinical potential. We used microarrays to identify early biomarkers that distinguish ischemic from nephrotoxic ARF, or biomarkers that detect both injury types. We compared rat kidney transcriptomes 2 and 8 hours after ischemia/reperfusion and after mercuric chloride. Quality control and statistical analyses were necessary to normalize microarrays from different lots, eliminate outliers, and exclude unaltered genes. Principal component analysis revealed distinct ischemic and nephrotoxic trajectories, and clear array groupings. Therefore, we used supervised analysis, ttests and fold changes, to compile gene lists for each group, exclusive or non-exclusive, alone or in combination. There was little network connectivity, even in the largest group. Some microarray-identified genes were validated by TaqMan assay, ruling out artifacts. Western blotting confirmed that HO-1 and ATF3 proteins were upregulated; however, unexpectedly, their localization changed within the kidney. HO-1 staining shifted from cortical (early) to outer stripe of the outer medulla (late), primarily in detaching cells, after mercuric chloride, but not ischemia/reperfusion. ATF3 staining was similar, but with additional early transient expression in the outer stripe after ischemia/reperfusion. We conclude that microarray-identified genes must be evaluated not only for protein levels, but also for anatomical distribution among different zones, nephron segments, or cell types. Although protein detection reagents are limited, microarray data lay a rich foundation to explore biomarkers, therapeutics, and pathophysiology of ARF.