In mammals mitochondria play a critical role in the activation of programmed cell death (PCD). One mechanism by which mitochondria can commit a cell to death is by translocating cytochrome c into the cytosol where it activates cell death caspases. However, release of cytochrome c does not appear to be a feature of caspase activation in nematodes or insects, similarly, there is no evidence for cytochrome c release during the caspase-independent PCD that can occur in Dictyostelium cells. In an attempt to understand the underlying regulation of PCD in plants we investigated if mitochondrial components were released into the cytosol when plant cells are induced to undergo PCD. PCD was triggered in cucumber cotyledons by subjecting them to a short 55°C heat treatment. This heat treatment has previously been shown to trigger PCD in other plant species and cell death was confirmed in cucumber using morphological (cellular condensation) and molecular (DNA ‘laddering’) markers of PCD. We present evidence that, unlike Dictyostelium and invertebrate PCDs, cytochrome c release is an early event in plant PCD. The mitochondrial release of cytochrome c following a PCD-inducing stimulus in both plants and mammals suggests the pathways have been conserved during evolution, having been derived from ancestral unicellular death programmes.