A and alpha cells of the yeast Saccharomyces cerevisiae exhibit an axial budding pattern, whereas a/alpha diploid cells exhibit a bipolar pattern. Mutations in BUD3, BUD4, and AXL1 cause a and alpha cells to exhibit the bipolar pattern, indicating that these genes are necessary to specify the axial budding pattern (Chant, J., and I. Herskowitz. 1991. Cell. 65:1203-1212; Fujita, A., C. Oka, Y. Arikawa, T. Katagi, A. Tonouchi, S. Kuhara, and Y. Misumi. 1994. Nature (Lond.). 372:567-570). We cloned and sequenced BUD4, which codes for a large, novel protein (Bud4p) with a potential GTP-binding motif. Bud4p is expressed and localized to the mother/bud neck in all cell types. Most mitotic cells contain two apparent rings of Bud4 immunoreactive staining, as observed for Bud3p (Chant, J., M. Mischke, E. Mitchell, I. Herskowitz, and J.R. Pringle. 1995. J. Cell Biol. 129: 767-778). Early G1 cells contain a single ring of Bud4p immunoreactive staining, whereas cells at START and in S phase lack these rings. The level of Bud4p is also regulated in a cell cycle-dependent manner. Bud4p is inefficiently localized in bud3 mutants and after a temperature shift of a temperature-sensitive mutant, cdc12, defective in the neck filaments. These observations suggest that Bud4p and Bud3p cooperate to recognize a spatial landmark (the neck filaments) during mitosis and support the hypothesis that they subsequently become a landmark for establishing the axial budding pattern in G1.