Barth syndrome is an X-linked mitochondria disorder with skeleton myopathy, cardiomyopathy and neutropenia, resulting from the tafazzin mutation. As demonstrated previously [Xu et al.: PNAS 103:11584, 2006], the mutation of full length Drosophila tafazzin has led to deficiency of cardiolipin and abnormality of phopholipids in the Drosophila animal model. We have further used recombinant baculovirus-Sf9 insect cell system to identify the biochemical reaction of a full length mitochondrial membrane-bound Drosophila tafazzin protein and related isoforms. Tafazzin expression induced a new enzymatic function in Sf9 cell mitochondria, namely 1-palmitoyl-2-[14C]-linoleoyl-phosphatidylcholine: monolysocardiolipin linoleoyltransferase (Xu et al.: JBC 2007). The highest rate was for the phosphatidylcholine-cardiolipin transacylation between various phospholipid-lysophospholipid pairs. Transacylation activities were about ten-fold higher for linoleoyl groups than for oleoyl groups, and negligible for arachidonoyl groups. The reverse reaction was also evident by transfering acyl groups from phospholipids to 1-[14C]-palmitoyl-2-lyso-phosphatidylcholine. The purified tafazzin protein in mitochodnria using GST-tag affinity column determined by Western blot and silver staining, catalyzed both forward and reverse transacylations between cardiolipin and phosphatidylcholine, but was unable to utilize acyl-CoA as substrates. The data show that Drosophila tafazzin is a CoA-independent, acyl-specific phospholipid transacylase with substrate preference for cardiolipin and phosphatidylcholine.