Previous in vitro studies indicated that aldosterone nongenomically phosphorylates epidermal growth factor receptor (EGFR) through activation of upstream signals, heat shock protein 90 β (Hsp90 β ), and cytosolic (c)-Src kinase. We demonstrated that aldosterone rapidly elevates EGFR phosphorylation in rat kidney. There are no in vivo data regarding renal Hsp90( α and β ) and phosphorylated (p)c-Src protein expressions. The present study further investigates the expressions of these proteins. Male Wistar rats were intraperitoneally injected with normal saline solution or aldosterone (Aldo: 150 μ g/kg BW). After 30 minutes, abundances and localizations of these proteins were determined. Aldosterone enhanced renal Hsp90 β protein abundance (P < 0.001), but Hsp90 α and pc-Src protein levels remained unaltered. Expression of Hsp90( α and β ) was induced prominently in the proximal convoluted tubules (PCTs). Activation of Hsp90 α was observed in vascular and outer medulla regions, whereas Hsp90 β was induced in the cortex. Immunoreactivity of pc-Src was elevated in PCT with obvious staining at the luminal membrane. This in vivo study is the first to demonstrate that aldosterone nongenomically elevates Hsp90( α and β ) protein expressions in rat kidney. Aldosterone had no effect on pc-Src protein levels but modulated localization. These results indicate that aldosterone regulates upstream mediators of EGFR transactivation in vivo.