The two major zona pellucida proteins of the zebrafish chorion, Zp2 and Zp3, are encoded by multicopy genes arranged in tandem arrays on chromosomes 20 and 2, respectively. Expression of these zp genes in zebrafish is oocyte specific, and we report herein that their activity in developing oocytes is dependent on conserved CCAAT box sites in their promoters. A 140-bp region immediately upstream of the transcription initiation site (position 1) of the zp2 genes has been homogenized by gene conversion and contains a single CCAAT box located at −138 that is necessary for promoter activity in oocytes residing in stage I and early stage II ovarian follicles as determined by microinjection of promoter constructs linked to a luciferase reporter gene. The zp3 gene promoters have two inverted CCAAT boxes located in a region of shared homology within the initial 175 nucleotides. Serial deletion of these sites resulted in incremental decreases in luciferase activity. Double-stranded oligonucleotides containing CCAAT box sequences from both genes formed CCAAT box-specific complexes with ovarian follicle extracts in an electrophoretic mobility shift assay. We also found that the expression of the separate zebrafish zp3b gene, more closely related to two oocyte-expressed medaka zpc genes than to the tandemly arrayed zebrafish zp3 genes, is not CCAAT box dependent. The significance that these results have in furthering our understanding of the regulation of zebrafish zp gene evolution and regulation is discussed.