Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT) Of those techniques, the NOS-catalyzed [C-14-]-L-arginine to [C-14-]-L-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent [1] Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography Results obtained from these samples showed no radioactive band for L-citrulline However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [C-14]-L-arginine to [C-14]-urea The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays (C) 2009 Elsevier Inc All rights reserved