Although triacylglycerol stores play the critical role in an organism's ability to withstand fuel deprivation and are strongly associated with such disorders as diabetes, obesity, and atherosclerotic heart disease, information concerning the enzymes of triacylglycerol synthesis, their regulation by hormones, nutrients, and physiological conditions, their mechanisms of action, and the roles of specific isoforms has been limited by a lack of cloned cDNAs and purified proteins. Fortunately, molecular tools for several key enzymes in the synthetic pathway are becoming available. This review summarizes recent studies of these enzymes, their regulation under varying physiological conditions, their purported roles in synthesis of triacylglycerol and related glycerolipids, the possible functions of different isoenzymes, and the evidence for specialized cellular pools of triacylglycerol and glycerolipid intermediates.