Abstract Amelogenin phosphorylation is critical to the development of enamel. Individuals with amelogenesis imperfecta (AI) exhibit enamel hypoplasia and overall structure and integrity of enamel is weakened. AMELX-KI mice lack amelogenin phosphorylation and exhibit features of AI. Studies are still needed to understand the mechanisms of amelogenin phosphorylation. Using single cell RNA sequencing (scRNA-seq) and RT-qPCR we found acid phosphatase 4 (ACP4) to be significantly upregulated in the enamel organ of AMELX-KI mice. Published articles have shown that ACP4 is a transmembrane protein found in secretory ameloblasts, particularly in the Tomes’s processes. ACP4 was found in the Tomes’s processes of wildtype (WT) mice with higher expression in KI secretory ameloblasts using immunohistochemistry staining. Using western blot analysis, we found ACP4 expression in the enamel organ layer in both WT and AMELX-KI mice. Publications suggest that ACP4 is upregulated in a local acidic environment with higher expression in the secretory stages than in maturation stages. This supports our findings that higher ACP4 expression found in AMELX-KI mice may correlate to their lower pH levels in secretory stages. These findings have led to the hypotheses that interactions of phosphorylated amelogenin with ACP4 are essential for proper enamel formation and that changes in pH can affect these interactions. We will investigate protein-protein interactions between phosphorylated amelogenin and ACP4 using in situ proximity ligation (isPLA) and co-immunoprecipitation (Co-IP) assays. We will assess distribution and quantities of ACP4 and its relationships with amelogenin in WT and KI secretory ameloblasts using high resolution confocal microscopy. Finally, we will determine the effects of pH on phosphorylated AMELX and ACP4 interactions using isPLA on ameloblast lineage cells transfected with ACP4 and use acid phosphatase assay to determine activity. These studies will support the role of ACP4 expression in enamel development and will provide in-depth knowledge towards mechanisms of amelogenin phosphorylation.