A genome-wide screen of 4168 homozygous diploid yeast deletion strains has been performed to identify nonessential genes that participate in the bipolar budding pattern. By examining bud scar patterns representing the sites of previous cell divisions, 127 mutants representing three different phenotypes were found: unipolar, axial-like, and random. From this screen, 11 functional classes of known genes were identified, including those involved in actin-cytoskeleton organization, general bud site selection, cell polarity, vesicular transport, cell wall synthesis, protein modification, transcription, nuclear function, translation, and other functions. Four characterized genes that were not known previously to participate in bud site selection were also found to be important for the haploid axial budding pattern. In addition to known genes, we found 22 novel genes (20 are designated BUD13-BUD32) important for bud site selection. Deletion of one resulted in unipolar budding exclusively from the proximal pole, suggesting that this gene plays an important role in diploid distal budding. Mutations in 20 other novelBUD genes produced a random budding phenotype and one produced an axial-like budding defect. Several of the novel Bud proteins were fused to green fluorescence protein; two proteins were found to localize to sites of polarized cell growth (i.e., the bud tip in small budded cells and the neck in cells undergoing cytokinesis), similar to that postulated for the bipolar signals and proteins that target cell division site tags to their proper location in the cell. Four others localized to the nucleus, suggesting that they play a role in gene expression. The bipolar distal marker Bud8 was localized in a number of mutants; many showed an altered Bud8-green fluorescence protein localization pattern. Through the genome-wide identification and analysis of different mutants involved in bipolar bud site selection, an integrated pathway for this process is presented in which proximal and distal bud site selection tags are synthesized and localized at their appropriate poles, thereby directing growth at those sites. Genome-wide screens of defined collections of mutants hold significant promise for dissecting many biological processes in yeast.