Peroxidation of n‐6 and n‐3 PUFAs generates toxic aldehydes 4‐ hydroxynonenal (HNE) and 4‐hydroxyhexenal (HHE), respectively. The high content of PUFAs and ROS in mitochondria make them primary sources and targets of these aldehydes, particularly in oxidative tissues such as heart. The mitochondrial enzyme aldehyde dehydrogenase‐2 (ALDH2) is responsible, in part, for neutralizing these aldehydes. To determine whether HNE and HHE differ in their effect on mitochondrial function in heart, and if activation of ALDH2 can suppress these effects, mitochondria were isolated from rat heart (N=8) and treated with vehicle (CON), HNE or HHE in the presence or absence of Alda‐1 (100 μM; ALDH2 agonist). Maximal ADP‐stimulated O2 consumption (maxO2) supported by glutamate+malate (GM), pyruvate+malate and succinate was decreased ~70% by HNE in concentration (10–80 μM) and time‐dependent manner (P<0.05). maxO2 was not affected by HHE in identical treatments. Activation of ALDH2 with Alda‐1 attenuated the HNE‐mediated inhibition of GM‐supported maxO2 (P<0.05). Mitochondrial calcium tolerance (mCa2+) was reduced by HNE (−28.2 %) and HHE (−25.1 %) vs. CON (P<0.05), although much more HHE than HNE was required. Activation of ALDH2 did not restore mCa2+ to CON levels with either aldehyde. Collectively, these findings suggest that aldehydes derived from n‐6 PUFAs are more toxic to mitochondria than aldehydes derived from n‐3 PUFAs, and that activation of ALDH2 remains a provocative therapeutic target to suppress the cytotoxic action of HNE. (Supported by NIH grant HL098780 to EJA)