This study investigates the mechanism of hormonal regulation of p53 gene expression in MCF-7 human breast cancer cells. 17β-Estradiol (E2) induced a 2-fold increase in p53 mRNA levels and a 2- to 3-fold increase in p53 protein. Analysis of the p53 gene promoter has identified a minimal E2-responsive region at −106 to −40, and mutation/deletion analysis of the promoter showed that motifs that bind CCAAT-binding transcription factor-1 (CTF-1) and nuclear factor κB (NFκB) proteins are required for hormone responsiveness. The p65 subunit of NFκB was identified in both nuclear and cytosolic fractions of untreated MCF-7 cells; however, formation of the nuclear NFκB complex was E2 independent. Hormonal activation of constructs containing p53 promoter inserts (−106 to −40) and the GAL4-p65 fusion proteins was inhibited by the intracellular Ca²⁺ ion chelator EGTA-AM and Ca²⁺/calmodulin-dependent protein kinase (CaMK) inhibitor KN-93. Constitutively active CaMKIV but not CaMKI activated p65, and treatment of MCF-7 cells with E2 induced phosphorylation of CaMKIV but not CaMKI. The results indicate that hormonal activation of p53 though nongenomic pathways was CaMKIV-dependent and involved cooperative p65-CTF-1 interactions.