Acyl-CoA:lysocardiolipin acyltransferase-1 (ALCAT1) catalyzes acylation of lysocardiolipin back to cardiolipin, an important step involved in cardiolipin remodeling. The present study reports the catalytic properties of ALCAT1 in vitro and its regulation by thyroid hormone status in mouse liver and heart. The recombinant ALCAT1 expressed in Sf9 cells preferred basic pH conditions, and did not require divalent cations or the integrity of subcellular membrane for its enzymatic activity. The recombinant ALCAT1 was potently inhibited by ADP and ATP, but not by adenosine nucleotide analogues or other nucleotides such as UTP and GTP, suggesting that the ALCAT1 does not require ATP hydrolysis for its enzyme activity. In addition to cardiolipin, ALCAT1 also catalyzed acylation of other members of polyglycerophospholipid family, including phosphotidylglycerol (PG), an precursor for cardiolipin synthesis, and bis(monoacylglycero)phosphate, a structural isomer of PG and a metabolic intermediate of cardiolipin. These findings suggest a role of ALCAT1 in the remodeling of other polyglycerophospholipids. In support of a regulatory role of ALCAT1 in cardiolipin remodeling in response to oxidative stress, ALCAT1 expression in liver and heart was significantly down-regulated in mice with hypothyroidism, and up-regulated in mice treated with thyroid hormone which is known to stimulate mitochondrial activity, oxidative stress, and cardiolipin remodeling.