The zona pellucida (ZP) is an extracellular glycoprotein coat that is deposited around the oocyte during folliculogenesis and performs several functions that relate to fertilisation and preimplantion development. In eutherian mammals it consists of three major glycoproteins – ZPA, ZPB, and ZPC – but little is known about its molecular constitution in marsupials. We have isolated the cDNA encoding the ZPA homologue in two distantly related marsupial series: the possum, Trichosurus vulpecula (a phalangerid) and the dunnart Sminthopsis crassicaudata (a dasyurid). The two cDNA sequences were 86% identical and showed extensive regions of homology to eutherian ZPA proteins, particularly in the central region of the molecule. Many other features of the ZPA protein, except the positioning of the N-linked glycosylation sites, were also conserved between marsupials and eutherians. ZPA expression was shown to occur maximally in the cytoplasm of the oocyte primary follicles with a little, but significant, expression in oocytes of both primordial follicles and in the cytoplasm of the oocyte in follicles with an antral cavity. No expression was seen in surrounding follicle or granulosa cells