Various stressful conditions such as ischemia in cold cardioplegic solutions and reperfusion occur during heart transplantation. Since ATP production is essential for the maintenance of contractile activity, mitochondrial function may be a mediator of ischemia and ischemia/reperfusion (I/R) injury. We aimed at testing the ability of two distinct cardioplegic solutions, Celsior (Cs) and Histidine Buffer (HBS), to protect rat heart mitochondria (HM) function during ischemia alone or ischemia followed by reperfusion. A standard Krebs-Henseleit solution (KH) was used as "negative" control. Male and Female Wistar rats were divided into control (Ctrl), reperfusion control (Ctrl_R), ischemia and I/R groups. Ischemia and I/R were divided into three subgroups depending on the cardioplegic solution used (Cs, HBS or KH) and subjected to 4-or 6-h ischemia alone or followed by reperfusion. HM respiration and transmembrane electric potential (Deltapsi) were measured with oxygen and TPP(+)-selective electrodes, respectively. Mitochondrial electron microscopy and detection of protein carbonyl groups content were also performed. After ischemic heart preservation, mitochondrial respiration and Deltapsi were not significantly affected except for the respiratory control ratio (RCR). After I/R, state 3 respiration, RCR and Deltapsi were decreased, especially in HM from male and for complex I substrates (CxI). HM preserved in HBS had less membrane disruption, segregation or disintegration. We conclude that (a) female HM were less sensitive to I/R, (b) CxI was particularly affected by I/R, (c) two cardioplegic solutions tested act in different mitochondrial targets preventing mitochondrial collapse.