In heterothallic filamentous ascomycetes, two nuclei of opposite mating type must recognize one another in a plurinucleate cell to form a pair prior to karyogamy. In pseudohomothallic species, two nuclei of opposite mating type must also pair after meiosis to form a binucleate spore. We have examined the cytoskeletal involvement in nuclear pairings by immunofluorescence and drug disruption, using heterothallic and pseudohomothallic species, as well as species without defined mating type (homothallic). Nuclei of species with defined mating type have spindle pole bodies which react with chromatin stains; those of homothallic species do not. The reactivity is seen only in interphase, not during nuclear divisions; thus, the DNA concerned is nuclear and not organellar. From light and immunofluorescence microscopy, the DNA is located at the nuclear face of the spindle pole body (SPB). We suggest that the DNA-SPB association may be involved in the recognition of self and nonself between nuclei of opposite mating types. Nuclei which cooperate in cell formation during ascus development or sporulation are placed in close proximity by the arrangement of spindles during the division preceding cell formation; after division, each nuclear pair remains linked by intertwined microtubule asters. Nuclear pairs must migrate before binucleate spore formation. Drug disruptions established that actin-myosin interaction was the most important cytoskeletal factor in normal spore production. The ascomycete SPB shows unexpected flexibility in form and location during development. Prior to sporulation the outer plaque shows extensive modification in size and orientation. The modified portion detaches from the nucleus and acts as a cortical microtubule organizing center, while the rest of the spindle pole body remains at the nucleus.