YAL044, a gene on the left arm of Saccharomyces cerevisiae chromosome one, is shown to code for the Hprotein subunit of the multienzyme glycine cleavage system. The gene designation has therefore been changed to GCV3, reflecting its role in the glycine cleavage system. GCV3 encodes a 177-residue protein with a putative mitochondrial targeting signal at its amino terminus. Targeted gene replacement shows that GCV3 is not required for growth on minimal medium; however, it is essential when glycine serves as the sole nitrogen source. Studies of GCV3 expression revealed that it is highly regulated. Supplementation of minimal medium with glycine, the glycine cleavage system's substrate, induced expression at least 30-fold. In contrast, and consistent with the cleavage of glycine providing activated single-carbon units, the addition of the metabolic end products that require activated single-carbon units repressed expression about 10-fold. Finally, like many amino acid biosynthetic genes, GCV3 is subject to regulation by the general amino acid control system. The glycine cleavage system, a multienzyme complex consisting of four different subunits (P-, H-, T-, and L-proteins), catalyzes the oxidative cleavage of glycine into CO 2 and NH 3 .