To clarify the relationship of gap junction formation to phosphorylation of connexin43 (Cx43) in mouse preimplantation embryos, immunofluorescence and Western blot analysis were conducted. Immunofluorescence showed Cx43 positive spots first at the mid-eight-cell stage (6 hr postdivision to the eight-cell stage). The number of spots increased from 6 to 15 hr postdivision to the eight-cell stage. Western blot analysis suggested Cx43 to possibly be present in the nonphosphorylated form at the mid-four-cell stage (6 hr postdivision to the four-cell stage), and phosphorylated Cx43 to increase from the mid-eight-cell stage (6 hr post-division to the eight-cell stage) onward. Dibutyryl cAMP (dbcAMP), a protein kinase A (PKA) activator, added to the culture medium increased the phosphorylation of Cx43 and Cx43 positive spots. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, increased the phosphorylation of Cx43, but decreased Cx43 positive spots. These results suggest that the phosphorylation of Cx43, induced by different protein kinase, leads to a different effect on gap junction formation in mouse preimplantation embryos.