Abstract. The three dimensional organization of microtubules in mitotic spindles of the yeast Saccharomyces cerevisiae has been determined by computer-aided reconstruction from electron micrographs of serially cross-sectioned spindles. Fifteen spindles ranging in length from 0.6-9.4 Ixm have been analyzed. Ordered microtubule packing is absent in spindles up to 0.8 Ixm, but the total number of microtubules is sufficient to allow one microtubule per kinetochore with a few additional microtubules that may form an interpolar spindle. An obvious bundle of about eight interpolar microtubules was found in spindles 1.3-1.6 p~m long, and we suggest that the ~32 remaining microtubules act as kinetochore fibers. The relative lengths of the microtubules in these spindles suggest that they may be in an early stage of anaphase, even though these spindles are all situated in the mother cell, not in the isthmus between mother and bud. None of the reconstructed spindles exhibited the uniform populations of kinetochore microtubules characteristic of metaphase. Long spindles (2.7-9.4 p,m), presumably in anaphase B, contained short remnants of a few presumed kinetochore microtubules clustered near the poles and a few long microtubules extending from each pole toward the spindle midplane, where they interdigitated with their counterparts from the other pole. Interpretation of these reconstructed spindles offers some insights into the mechanisms of mitosis in this yeast. THE structure of the mitotic spindle and its function have been studied in a wide variety of organisms, yielding a few global observations about spindle structure. In general, spindles are organized from two spindle poles, each of which nucleates several classes of microtubules. These classes include astral, kinetochore, and interpolar spindle microtubules. The astral microtubules are not part of the spindle per se, but can be involved in the orientation, and perhaps the elongation of the spindle. Kinetochore microtubules connect the chromosomes to the spindle poles and are involved in chromosome movements. The interpolar spindle microtubules extend from each spindle pole, interdigitate with their counterparts from the other pole, and are involved in separating the poles with their attached chromosomes during anaphase B. This generic view of spindle organization is thought by many to hold true for most spindles, including