Protein import into isolated tobacco mitochondria was investigated using mitochondria from leaves harvested at different times of the day and night. Efficient import was only detected with mitochondria isolated from leaves harvested during the dark period of the growth cycle, only low levels of import were detected from leaves harvested during the light period. However, this temporal difference seen in import did not appear to be circadian in nature. This implies that the protein import process in mitochondria isolated from leaves is not constitutive. This has important implications for targeting specificity studies performed in transgenic plants, as unless the plants are tested at the time when import is occurring, the true in vivo targeting abilities of chimeric constructs will not be measured.