Other studies have shown that caffeine accelerates telencephalic vesicle evagination in early post-implantation mouse embryos. The present study examines the effect of caffeine on gene modulation in post-implantation mouse embryos. Using mRNA differential display, we observed that caffeine increased gene expression of the regulatory subunit (RI alpha) of cAMP-dependent protein kinase (PKA). RT--PCR analysis confirmed an increase in expression of this gene in caffeine-exposed embryos when compared with saline-treated controls. Using a fluorescent substrate of PKA, we found that PKA activity in the presence of cAMP was lower in caffeine-treated embryos than in controls. Treatment with H89 and PKI(12-24)amide, two inhibitors of PKA activity, mimicked the effects of caffeine on telencephalic vesicle formation. Together these data suggest that in early post-implantation mouse embryos caffeine modulates gene expression of the RI alpha subunit of PKA and that caffeine-induced inhibition of PKA activity plays a role in early telencephalic evagination.