AbstractThere are conflicting data about localization of poly(ADP‐ribose)polymerase‐1 and its product poly(ADP‐ribose) in mitochondria. To finally clarify the discussion, we investigated with biochemical and cell biological methods the potential presence of poly(ADP‐ribose) polymerase‐1 in these organelles. Our data show that endogenous and overexpressed poly(ADP‐ribose)polymerase 1 is only localized to the nucleus with a clear exclusion of cytosolic compartments. In addition, highly purified mitochondria devoid of nuclear contaminations do not contain poly(ADP‐ribose)polymerase‐1. Although no poly(ADP‐ribose)polymerase‐1 enzyme is detectable in mitochondria, a shorter variant of its product poly(ADP‐ribose) is present, associated specifically with a small subset of mitochondrial proteins as revealed by immunoprecipitation and protein fingerprint analysis. These proteins are located at key‐points of the Krebs‐cycle, are chaperones involved in mitochondrial functionality and quality‐control, and are RNA‐binding proteins important for transcript stability, respectively. Of note, despite the fact that especially poly(ADP‐ribose)polymerase‐1 is its own major target for modification, we could not detect this enzyme by mass spectrometry in these organelles. These data suggests a new way of targeted nuclear‐mitochondrial signaling, mediated by nuclear poly(ADP‐ribosyl)ation dependent on poly(ADP‐ribose)polymerase‐1.