The gene encoding Xenopus B-Myb (XB-Myb), a protein structurally related to the nuclear protooncogene product c-Myb, is expressed in early Xenopus embryogenesis. We report on developmental alterations in the nucleocytoplasmic distribution and phosphorylation of XB-Myb in Xenopus oocytes and embryos, as well as on a negative regulatory role of the carboxyl terminus in sequence specific DNA binding. In growing oocytes and early embryonic stages the protein is primarily located in the nucleus; in the full-grown oocyte, however, it remains sequestered in the cytoplasmic compartment. Upon meiotic maturation of the oocyte, XB-Myb becomes hyperphosphorylated. Oocyte/egg isolates of XB-Myb are inhibited in their specific DNA binding activity; truncation of the carboxyl terminal region relieves this block in nucleic acid recognition. Furthermore, we have used overexpression of XB-Myb in Xenopus embryos by means of mRNA injection as an assay for gene function in vivo. Overexpression of full-length XB-Myb, not of the carboxyl terminal deletion mutant, results in an altered morphology of lateral plate mesoderm.