In rodent models of obstructive nephropathy, exogenous EGF attenuates tubule cell death in rats and exacerbates cell death in mice. To identify species differences in EGFR regulation and signaling, cell lysates were prepared from rat, mouse and human proximal tubule cells (PTC) and compared by immunoblot analysis for expression and phosphorylation of Src and EGFR. Frozen kidney tissue was also analyzed. Results indicate mouse PTC have constitutive Src- and EGFR-kinase activities not detected in rat or human PTC. Immunoblots of rat, mouse and human kidney homogenates confirmed this finding in vivo. Src-specific inhibitor PP2 and EGFR kinase inhibitor AG1478 decreased EGF-induced apoptosis in mouse PTC by 74% (p < 0.001) and 70% (p < 0.001), respectively. Expression of a constitutive Src mutant cDNA in rat PTC rendered cells susceptible to EGF-induced death. EGF decreased stretch-induced apoptosis by 66% (p < 0.001) relative to vehicle control in human PTC, similar to rat PTC response. We conclude that elevated Src activity in mouse tubular cells alters down-stream EGFR signaling and increases susceptibility to EGF-induced cell death. The unexpected finding that a therapeutic agent (EGF) in rats is detrimental in mice underscores the importance of determining which animal best represents the response of human kidneys to a given agent.