Double knockout (DKO) of the small heat shock proteins CRYAB and HSPB2 increases necrosis and apoptosis induced by ischemia/reperfusion (I/R) in vitro, but the mechanisms involved are unknown. We examined [Ca2+]i during metabolic inhibition (MI) changes in [Ca2+]m induced by exposure to elevated [Ca2+]i, and whether mitochondria in isolated DKO ventricular myocytes (VM) are more susceptible than wild type (WT) to induction of the mitochondrial permeability transition (MPT). The rise in [Ca2+]i in DKO myocytes during metabolic inhibition (MI) was less than in WT, and ouabain caused a greater increase in [Ca2+]m in DKO than in WT. These findings suggested that Ca2+ uptake was increased in mitochondria in DKO myocytes. Measurements of Rhod 2 fluorescence during exposure of permeabilized VM to 1000 nM [Ca2+] for 5 min confirmed that DKO myocytes have enhanced mitochondrial Ca2+ uptake, and this difference between DKO and WT myocyte mitochondria was eliminated by inhibition of NO synthesis. MPT was induced more readily by ouabain, PAO, or TMRM in DKO myocytes than in WT. Thus, Ca2+ uptake by mitochondria is increased in DKO VM by a NO-dependent mechanism. This can predispose to the development of MPT, and increased VM injury during I/R. These findings indicate an important role of CRYAB and/or HSPB2 in mitochondrial function.