The fruit of the 'Hiratanenashi' persimmon (Diospyros kaki Thunb.) over-ripens on the tree by late November as a result of senescence processes. On-tree over-ripened fruits were harvested at this time, and the degree of fruit softening was measured. The 'softening degree' index ranged from 1.5 (firm to moderately soft) to 4 (very soft, or part of the peel ruptured). To clarify the fruit softening processes resulting from on-tree senescence, internal ethylene concentrations, gene expression of ethylene synthesis, and cell-wall-degrading enzymes were investigated in 'Hiratanenashi' persimmon. The internal ethylene concentration increased concomitantly with softening progress. The expression of two ACC synthase (ACS) genes, DkACS1 and DkACS2, and two ACC oxidase (ACO) genes, DkACO1 and DkACO2 were investigated using quantitative PCR. The ACS genes were rate limiting for ethylene production, but ACO genes were constitutive. Genes encoding three polygalacturonases (PG), two pectin methylesterases (PE), two β-galactosidases (Gal), and one α-arabinofuranosidase (Arf) were isolated from an expressed sequence tag (EST) library of on-tree softened pulp, and their expression was investigated. Among these eight isolated genes, the expression of DkPG1, DkPG2, DkGal1, and DkArf1 increased concomitantly with softening progress. In contrast, the expression of DkPG3, DkPE1, DkPE2, and DkGal2 was maintained at low levels throughout the softening progress. These results suggest that DkPG1, DkPG2, DkGal1, and DkArf1 play a coordinated role in on-tree fruit softening that follows PE action by senescence in 'Hiratanenashi'. Two of the PG genes are thought to contribute to a jelly- or soup-like texture at softening degree 4.