A new assay has been developed to measure mitochondrial ATP synthesis of cultured mammalian cells Cells in a microplate are exposed to streptolysin O to make plasma membranes permeable without damaging mitochondrial function and ATP synthesis is monitored by luciferase Addition of inhibitors of FoF1-ATP synthase (FoF1) respiratory chain TCA cycle and ATP/ADP translocator as well as knockdown of beta-subunit of FoF(1) resulted in loss of ATP synthesis Compared with the conventional procedures that need mitochondria fractionation and detergent this assay is simple sensitive and suitable for high-throughput analysis of genes and drugs that could affect mitochondrial functional integrity as represented by ATP synthesis activity (C) 2010 Elsevier Inc All rights reserved