The expression of two members of an important family of transcription factors, cAMP response element-binding protein (CREB) and cAMP-dependent transcription factor ATF1 (ATF1), is essential for normal preimplantation development. There is a high degree of functional similarity between these two transcription factors, and they can both homodimerize and heterodimerize with each other to form active transcription factors. CREB is present in all stages of mouse preimplantation embryo, and we show here that ATF1 is localized to the nucleus in all preimplantation stages. Activation of these transcription factors requires their phosphorylation, and this was only observed to occur for both transcription factors (serine 133 phosphorylation of CREB and serine 63 phosphorylation of ATF1) at the two-cell stage. Nuclear localization and phosphorylation of ATF1 were constitutive. The nuclear localization and phosphorylation of CREB showed a constitutive component that was further induced by the autocrine embryotropin Paf (1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Activation of CREB by Paf was independent of cAMP but was dependent on calcium, calmodulin, and calmodulin-dependent kinase activity. ATF1 nuclear localization was unaffected by inhibition of the calcium/calmodulin pathway. A complex pattern of expression of calmodulin-dependent kinases was observed throughout preimplantation development. At the two-cell stage, only mRNAs coding for calmodulin-dependent protein kinase kinase beta, calmodulin-dependent protein kinase II gamma, and calmodulin-dependent protein kinase IV were detected. A selective antagonist for calmodulin-dependent protein kinase kinase (STO-609) and calmodulin-dependent protein kinases I, II, and IV (KN-62) blocked the Paf-induced phosphorylation of CREB. The study demonstrates a role for trophic signaling and constitutive activation of two essential transcription factors at the time of zygotic genome activation.