The three-dimensional structure of the Saccharomyces cerevisie fatty acid synthase was computed from electron microscopy of stain images. The barrel-shaped structure (point group symmetry 32) has major and minor axes of approximately 245 x 220 A, respectively, and consists of two different subunits organized in an alpha6beta6 complex (Mr = 2.5 x 10(6)). Two sets of three beta subunits form triangle-shaped caps that enclose the ends of the barrel. The wall of the barrel appears to consist of three N-shaped alpha subunit pairs each with an over and underlying arch-shaped beta subunit. Inside the molecule there are three major interconnected cavities that are tilted approximately 20 degrees with respect to its major axis. An axle-shaped structure extends the length of the cavity on the 3-fold axis and is connected to the two ends of the barrel. The cavities are partially divided on the equator of the molecule by three spokes that extend from the axle on the 2-fold axis to the exterior wall. We propose that these six cavities constitute the six equivalent sites of fatty acid synthesis resulting in an extraordinary structure-function relationship with the 42 catalytic sites involved in fatty acid synthesis inside the molecule. The six cavities each have two funnel-shaped openings ( approximately 20 A in diameter) which may serve to permit the diffusion of substrates and products in and out of these functional units. The subunits appear to be arranged in a manner that affords extensive intermolecular interactions contributing to the stability of this macromolecular complex.