Actinidin is a cysteine protease found in Actinidia Lindl. (kiwifruit) species that affects the nutraceutical properties, processing characteristics and allergenicity of the fruit. Given the increased consumption of kiwifruit worldwide and the release of new varieties from different Actinidia species, the expression of actinidin mRNA and protein in a range of kiwifruit tissues was examined. Ten different actinidin mRNAs were identified encoding mature proteins of similar molecular weight (~24 kDa), but with predicted pIs ranging from acidic (pI 3.9) to basic (pI 9.3). In A. deliciosa ‘Hayward’ (green-fleshed kiwifruit) and A. chinensis ‘Hort16A’ and EM4 (gold-fleshed kiwifruit), actinidin mRNAs for acidic and basic proteins were expressed at comparable levels throughout ripening. Actinidin mRNA expression was highest in fruit at harvest, expression decreased as fruit ripened and was much lower in the core compared with outer pericarp tissue. Two-dimensional gel electrophoresis, combined with western analysis and liquid chromatography mass spectrometry (LC-MS) identified low levels of a novel basic actinidin protein in ripe A. deliciosa and A. chinensis fruit. Extremely high levels of an acidic actinidin protein were detected in A. deliciosa fruit and EM4, but this acidic protein appeared to be absent in ‘Hort16A’, the most important commercial cultivar of A. chinensis. Analyses on native gels indicated that both the basic and acidic actinidin isoforms in A. deliciosa were active cysteine proteases. Immunolocalisation showed that actinidin was present in small cells, but not large cells in the outer pericarp of mature A. deliciosa fruit at harvest. Within the small cells, actinidin was localised diffusely in the vacuole, associated with the plasma membrane, and in a layer in the plastids near starch granules. The presence of multiple forms of actinidin and varying protein levels in fruit will impact on the ability to breed new kiwifruit varieties with altered actinidin levels.