Actinidin, the 23.6 kDa cysteine proteinase of Chinese gooseberry (Actinidia chinensis), is present at high concentration in fruits. A fruit-specific cDNA library was established and screened by differential hybridisation and using a synthetic oligonucleotide. Two of ten actinidin clones identified were characterised by sequence analysis. The two very similar cDNAs code for proteins with approximately 90% sequence homology to the published amino acid sequence of actinidin, as well as an additional 25 amino acids following the mature carboxyl terminus. The larger clone in addition has coding potential for 57 residues of an amino-terminal extension with considerable homology to amino-terminal sequences of other cysteine proteinases. From size determinations of both mRNA (1.4 kb) and immunoprecipitated in vitro translation product (39 kDa) it was estimated that actinidin is synthesised as a precursor approximately 15 kDa larger than the mature protein. Features of the prosegment primary sequence are considered with regard to a possible mechanism of inactivation of the proteinase, by analogy with other proteolytic zymogens. The presence of three potential glycosylation sites, one within the carboxy-terminal and two in the amino-terminal extension are consistent with subcellular location of the enzyme within membrane-bound organelles. Results from a Southern blot show that actinidin is encoded by a multigene family of up to ten members. Actinidin gene expression, both at the level of mRNA and protein, is largely restricted to the fruit of A. chinensis, where the level of actinidin mRNA accumulates early during development.