The mitochondrial matrix contains endogenously biotinylated proteins. These proteins can cause unexpected background signal when biotin-avidin- or biotin-streptavidin-based detection systems are used in immunocytochemistry. Here we show that this reactivity can be deliberately exploited, using a simple anti-biotin reagent, to obtain strong and highly specific labeling of mitochondria by both light and electron microscopy. The signal is substantially stronger than when either avidin or streptavidin is used to detect the endogenous biotin. These results confirm the accessibility of protein-bound endogenous biotin to exogenous probes, and localize the biotinylated enzymes to the mitochondrial matrix.