In the yeast Saccharomyces cerevisiae, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), which convert glucose 6- phosphate plus UDP-glucose to trehalose, are part of the trehalose synthase complex. In addition to the TPS1 (previously also called GGS1, CIF1, BYP1, FDP1, GLC6, and TSS1) and TPS2 (also described as HOG2 and PFK3) gene products, this complex also contains a regulatory subunit encoded by TSL1. We have constructed a set of isogenic strains carrying all possible combinations of deletions of these three genes and of TPS3, a homologue of TSL1 identified by systematic sequencing. Deletion of TPS1 totally abolished TPS activity and measurable trehalose, whereas deletion of any of the other genes in most cases reduced both. Similarly, deletion of TPS2 completely abolished TPP activity, and deletion of any of the other genes resulted in a reduction of this activity. Therefore, it appears that all subunits are required for optimal enzymatic activity. Since we observed measurable trehalose in strains lacking all but the TPS1 gene, some phosphatase activity in addition to Tps2 can hydrolyze trehalose 6-phosphate. Deletion of TPS3, in particular in a tsl1Δ background, reduced both TPS and TPP activities and trehalose content. Deletion of TPS2, TSL1, or TPS3 and, in particular, of TSL1 plus TPS3 destabilized the trehalose synthase complex. We conclude that Tps3 is a fourth subunit of the complex with functions partially redundant to those of Tsl1. Among the four genes studied, TPS1 is necessary and sufficient for growth on glucose and fructose. Even when overproduced, none of the other subunits could take over this function of Tps1 despite the homology shared by all four proteins. A portion of Tps1 appears to occur in a form not bound by the complex. Whereas TPS activity in the complex is inhibited by P(i), P(i) stimulates the monomeric form of Tps1. We discuss the possible role of differentially regulated Tps1 in a complex-bound or monomeric form in light of the requirement of Tps1 for trehalose production and for growth on glucose and fructose.