The overwinter bud of the gentian plant, Gentiana triflora, is an organ that develops from the basal tuber in late spring and becomes dormant and freezing-tolerant for overwintering. Here, proteins specifically enriched in the overwinter buds were identified using two-dimensional polyacrylamide gel electrophoresis followed by amino acid sequencing. Enriched proteins included those so far identified as stress- and cold-inducible in other plants, i.e., two isoforms of ethylene-induced esterase, a dehydration-induced protein, glyoxalase I, thioredoxin peroxidase, two isoforms of ascorbate peroxidase and a membrane pore protein. Interestingly, the stress-related proteins had already accumulated in the overwinter buds under non-stress conditions in early summer. Accumulation of these proteins was also examined in a mutant gentian, which developed overwinter buds but lacked cold tolerance and died under conditions below 3°C. In this mutant, several proteins including one of the putative esterase, enolase and ascorbate peroxidase were absent or greatly decreased. Thus, it appears that these stress-related proteins are expressed under non-stress conditions to aquire cold tolerance before overwintering.