Association of arginase and ornithine transcarbamoylase (OTCase) has been proposed to play an essential role in the regulation of arginine metabolism in Saccharomyces cerevisiae (Wiame, J.-M. (1971) Curr. Top. Cell. Reg. 4, 1-39). In this report multienzyme complex formation is directly demonstrated in the presence of the active-site ligands for OTCase and arginase. Using equilibrium sedimentation, a dissociation constant for complex formation was determined to be 2.3 X 10(-8) M in the presence of ornithine and agmatine, active-site ligands for OTCase and arginase, respectively. A molecular stoichiometry in the complex of one molecule of OTCase to one molecule of arginase was verified using transmission electron microscopy. The dimensions of the complex were determined by negative staining and rotary and unidirectional shadowing techniques to be 102 A wide by 81 A high. These dimensions are quantitively consistent with dimensions of the individual enzymes (Duong, L. T., Eisenstein, E., Green, S. M., Ornberg, R. L., and Hensley, P. (1986) J. Biol. Chem. 261, 12807-12813). The enzymatic activity of OTCase is virtually completely inhibited when associated with arginase, reflecting the dramatic modulation of enzyme activity as a consequence of the acquisition of quaternary structure in this multienzyme complex.