We have previously shown that, in human cells, cruciform DNA-binding activity is due to 14-3-3 proteins (Todd, A., Cossons, N., Aitken, A., Price, G. B., and Zannis-Hadjopoulos, M. (1998) Biochemistry 37, 14317-14325). Here, wild-type and single- and double-knockout nuclear extracts from the 14-3-3 Saccharomyces cerevisiae homologues Bmh1p and Bmh2p were analyzed for similar cruciform-binding activities in relation to these proteins. The Bmh1p-Bmh2p heterodimer, present in the wild-type strain, bound efficiently to cruciform-containing DNA in a structure-specific manner because cruciform DNA efficiently competed with the formation of the complex, whereas linear DNA did not. In contrast, the band-shift ability of the Bmh1p-Bmh1p and Bmh2p-Bmh2p homodimers present in the bmh2(-) and bmh1(-) single-knockout cells, respectively, was reduced by approximately 93 and 82%, respectively. The 14-3-3 plant homologue GF14 was also able to bind to cruciform DNA, suggesting that cruciform-binding activity is a common feature of the family of 14-3-3 proteins across species. Bmh1p and Bmh2p were found to associate in vivo with the yeast autonomous replication sequence ARS307, as assayed by formaldehyde cross-linking, followed by immunoprecipitation with anti-Bmh1p/Bmh2p antibody and conventional PCR. In agreement with the finding of an association of Bmh1p and Bmh2p with ARS307, another immunoprecipitation experiment using 2D3, an anti-cruciform DNA monoclonal antibody, revealed the presence of cruciform-containing DNA in ARS307.