ABSTRACT A recessive mutation, hfd1—1, in strain SOS4 of Saccharomyces cerevisiae leads the mutant cells to produce predominantly two-spored asci. Light microscopical examination of Giemsastained cells revealed no significant differences in the meiotic figures between mutant and wild-type strains. However, only two of the four meiotic products in a developing ascus matured to ascospores in SOS4. Dyad analysis was carried out on an hfd1-1 mutant strain heterozygous for three markers, asp5, gal1 and arg4, which are closely linked to their centromeres, and for his4, which is loosely linked to its centromere. The twospored asci produced by the hfd1—1 mutant segregated dominant (+) and recessive (-) alleles of each marker in a 1:1 ratio; they generally contained one + and one - spore for any given marker. The occurrence of rare dyads with two + or two - spores can be explained quantitatively by recombination between the marker and its centromere. From the results of these cytological and genetical analyses, we infer that, in the mutant strain, one genome set is partitioned to each of the four second-meiotic division poles, but only two nonsister genomes are incorporated into mature spores. Thus, the hfd1—1 mutation in SOS4 blocks incorporation of two nonsister nuclei into mature ascospores, but does not block enclosure of the remaining two nonsister nuclei.