Treatment of PtK1 cells during metaphase with solutions containing hyperosmotic concentrations of sucrose resulted in an alteration of kinetochore structure and function in a concentration-dependent manner. This alteration in kinetochore morphology was shown to be rapidly reversible upon removal of the sucrose-containing tissue culture medium. A 10-min treatment with both 0.2 M and 0.4 M sucrose resulted in a concentration-dependent aggregation of spindle fibers into bundles, loss of trilaminar kinetochore morphology as judged by electron microscopy, and induction of anaphase B-like spindle elongation as previously described. Electron microscopy showed that a 10-min treatment of metaphase cells with hyperosmotic concentrations of sucrose changed the trilaminar kinetochore structure to one of a single lamina, with an amorphous, lightly staining material distally associated with it. Sucrose-induced bundles of microtubules could usually be seen embedded or tangentially associated with this material. Rate and extent of spindle elongation in sucrose-treated metaphase cells were greater in the higher concentrations of sucrose employed. The degree of microtubule bundling was also concentration dependent, with reduced bundling occurring at lower sucrose concentrations. Within 2 min after sucrose removal kinetochores returned to a bi- or trilaminar morphology with reduction in the amount of amorphous material. Reformation of the kinetochore trilaminar structure resembled that of the normal maturation process which occurs from prophase through anaphase. These rapid changes in kinetochore morphology following release from sucrose treatment were temporally associated with restoration of spindle function and suggested that kinetochore integrity was necessary for the expression of spindle forces responsible for spindle shortening. These forces are probably generated or transduced by the continuum formed between the two spindle poles, the kinetochore microtubules, and the sister chromatids.