A study involving markers of subcellular and submitochondrial fractions, gradient centrifugation, latency measurements and extraction with digitonin, demonstrates the association of a specific ADP-ribose pyrophosphatase with rat liver mitochondria and its localization in the matrix space. The enzyme hydrolyses ADP-ribose to AMP, with a Km of 2-3 microM. The results support the occurrence of a specific turnover pathway for free ADP-ribose and its relevance in mitochondria.