C/EBPalpha is implicated to regulate mouse amelogenin gene expression during tooth enamel formation in vitro. Because enamel formation occurs during postnatal development and C/EBPalpha-deficient mice die at birth, we used the Cre/loxP recombination system to characterize amelogenin expression in C/EBPalpha conditional knock-out mice. Mice carrying the Cre transgene under the control of the human keratin-14 promoter show robust Cre expression in the ameloblast cell lineage. Mating between mice bearing the floxed C/EBPalpha allele with keratin-14-Cre mice generate C/EBPalpha conditional knock-out mice. Real-time PCR analysis shows that removal of one C/EBPalpha allele from the molar enamel epithelial organ of 3-day postnatal mice results in dramatic decrease in endogenous C/EBPalpha mRNA levels and coordinately altered amelogenin mRNA abundance. Conditional deletion of both C/EBPalpha alleles further diminishes C/EBPalpha mRNA levels; however, rather than ablating amelogenin expression, we observe wild-type amelogenin mRNA abundance levels. We examined C/EBPbeta and nuclear factor YA expression, two transcription factors that had previously been shown to modestly participate in amelogenin expression, in vitro but found no significant changes in either of their mRNA abundance levels comparing conditional knock-out mice with wild-type counterparts. Although the abundance of C/EBPdelta is also unchanged in C/EBPalpha conditional knock-out mice, in vitro we find that C/EBPdelta activates the mouse amelogenin promoter and synergistically cooperates with nuclear factor Y, suggesting that C/EBPdelta can functionally substitute for C/EBPalpha to produce an enamel matrix competent to direct biomineralization.