The peptidyl-prolyl isomerase FKBP12 was originally identified as the cytosolic receptor for the immunosuppressive drugs FK506 and rapamycin. Although peptidyl-prolyl isomerases have been implicated in catalyzing protein folding, the cellular functions of FKBP12 in Sacharomyces cerevisiae remained unclear. To identify in vivo substrates of FKBP12, we used the yeast two-hybrid system, and found that FKBP12 physically interacts with the enzyme aspartokinase, which catalyzes an intermediate step in threonine and methionine biosynthesis. Aspartokinase also binds to an FKBP12-affigel affinity matrix in vitro, and this interaction is disrupted by drugs that bind the FKBP12 active site. In addition, mutations in FKBP12 surface and active site residues perturb the FKBP12-aspartokinase complex. FKBP12 mutants are viable and are not methionine or threonine auxotrophs, thus FKBP12 is not essential for aspartokinase activity. Control of the metabolic flow in this pathway is mainly through feedback inhibition of aspartokinase activity by threonine. Genetic analyses show that FKBP12 modulates feedback-inhibition by hydroxynorvaline, a threonine analog, and mutations in the FKBP12 peptidyl-prolyl isomerase active site prevent complex formation and proper regulation of this inhibition. Biochemical experiments are in progress to determine if FKBP12 mutants have increased aspartokinase activity in the presence of threonine or hydroxynorvaline.