Carrot cells cultured in vitro in a medium supplemented with 2,4-D (2,4-dichlorophenoxyacetic acid) proliferate as unorganized cell clusters. Upon removal of 2,4-D from the culture medium, these cells undergo somatic embryo formation through globular, heart, and torpedo stages. Since the proximal -255 bp upstream region of the rolC gene of the Ri plasmid confers somatic embryogenesis-related activation on the uidA gene in transgenic carrot cell culture, we investigated the interaction of nuclear proteins with the proximal -255bp upstream sequences to characterize the mechanism of somatic embryogenesis-related activation. Gel retardation experiments revealed that there were several different profiles of the relative levels of DNA binding activities in nuclear protein extracts from calli, PEMs (proembryogenic masses), globular embryos, and heart/torpedo embryos. The binding activity associated with a fragment (-203 bp to -92 bp) of one protein (BI) was most abundant in globular embryos. Another DNA binding protein (AII) showed the highest DNA binding activity in calli, but had low binding activities in PEMs and globular embryos. Such an altered pattern of DNA binding activities of nuclear proteins may contribute to somatic embryogenesis-related activation of the rolC promoter. Competition experiments with oligonucleotides revealed that the BI protein interacts with AT-rich sequences.