Zygote formation occurs through tightly coordinated cell and nuclear fusion events. Genetic evidence suggests that the FUS2 gene product promotes cell fusion during zygote formation in Saccharomyces cerevisiae, functioning with the Fus1 plasma membrane protein at or before cell wall and plasma membrane fusion. Here we report the sequence of the FUS2 gene, localization of Fus2 protein, and show that fus1 and fus2 mutants have distinct defects in cell fusion. FUS2 encodes a unique open reading frame of 617 residues that only is expressed in haploid cells in response to mating pheromone. Consistent with a role in cell fusion, Fus2 protein localizes with discrete structures that could be of cytoskeletal or vesicular origin that accumulate at the tip of pheromone-induced shmoos and at the junction of paired cells in zygotes. Fus2 is predicted to be a coiled-coil protein and fractionates with a 100,000 g pellet, suggesting that it is associated with cytoskeleton, membranes, or other macromolecular structures. Fus2 may interact with structures involved in the alignment of the nuclei during cell fusion, because fus2 mutants have strong defects in karyogamy and fail to orient microtubules between parental nuclei in zygotes. In contrast, fus1 mutants show no karyogamy defects. These, and other results suggest that Fus2 defines a novel cell fusion function and subcellular structure that is also required for the alignment of parental nuclei before nuclear fusion.