The yeast fatty acid synthase is a multifunctional enzyme composed of two nonidentical subunits in an alpha 6 beta 6 complex that is active in synthesizing fatty acids. The seven catalytic activities required for fatty acid synthesis are divided between the alpha and beta subunits such that the alpha 6 beta 6 complex has six complements of each activity. It has been proposed that these are organized into six centers for fatty acid synthesis. There are different opinions regarding the operation of these centers in the alpha 6 beta 6 complex, on view being that they are functionally independent and the other proposes half-sites activity for the complex. We have attempted to distinguish between these proposals by the most direct method of active site titration, i.e., quantitation of fatty acyl product in the absence of turnover. This was accomplished by using p-nitrophenyl thioacetate and thiophenyl malonate (in place of the coenzyme A analogues) as substrates along with NADPH, thereby depriving the yeast synthase of coenzyme A required to release product as fatty acyl coenzyme A. The amount of fatty acyl product formed was quantitated by gas-liquid chromatography, as well as by direct estimation of radioactivity in the product when p-nitrophenyl thio [1-14C] acetate was used as a substrate. In both cases, a stoichiometry of close to six was found for mole of fatty acid synthesized per mole of alpha 6 beta 6 complex. This indicates that there are six functional centers for fatty acid synthesis in the multifunctional yeast alpha 6 beta 6 fatty acid synthase and that these centers operate independently.(ABSTRACT TRUNCATED AT 250 WORDS)