Membrane-permeable cAMP analogs or elevation of intracellular cAMP by cyclic nucleotide phosphodiesterase (PDE) inhibitors activates cAMP-dependent protein kinase. Biologically active phorbol esters or diacylglycerol activate the calcium-, phospholipid-dependent protein kinase, protein kinase C (PK-C). We report that membrane-permeable cAMP analogs, PDE inhibitors, biologically active phorbol esters, or a synthetic diacylglycerol inhibited cleavage of 1-cell mouse embryos to the 2-cell stage. The cAMP analogs and PDE inhibitors were effective only when added prior to S of the first cell cycle, whereas PK-C activators inhibited cleavage when added up until late G2/M. The PDE inhibitor Ro 20 1724/1 inhibited both DNA and protein synthesis in 1-cell embryos, whereas the phorbol ester, 12-O-tetradecanoyl-phorbol-13 acetate, or alpha-amanitin did not. In addition, 1-cell embryos prevented from cleaving by PDE inhibitors did not show specific changes in the pattern of protein phosphorylation associated with the 2-cell embryo, whereas such changes occurred in 1-cell embryos inhibited from cleaving with PK-C activators. Transcription in the 2-cell embryo results in the synthesis of a specific set of proteins, which is inhibited by alpha-amanitin. Although treatment of 1-cell embryos with aphidicolin or PK-C activators during G1 did not inhibit the synthesis of these proteins, treatment with cAMP analogs or PDE inhibitors during G1 inhibited the appearance of these proteins. These results are discussed in terms of how the synthesis of transcription-dependent proteins in the 2-cell embryo may be regulated by protein phosphorylation.