Biochemical changes in potato leaves during cold acclimation have been examined and compared between a frost-tolerant S. acauk and a frostsusceptible S. tuberosum species. Changes were also examined in S. tuberosum, S. acauk, and S. commersonii species when they were hardened at different temperatures to varying hardiness levels.During three weeks of stepwise cold accfimation, S. acauk increased frost hardiness from -6.0 C (killing temperature) to -9.0 C, whereas frost hardiness of S. tuberosum remained unchanged at -3.0 C. Decreases in DNA content on a dry weight basis in both species suggest that matured leaf cells accumulated more dry matter during acclimation. The advantage of using DNA as a reference for comparing metabolite changes during cold acclimation is discussed.Under the stepwise acclimating conditions, both species showed the same trends for increasing total sugar and starch with an insignificant decrease in leaf water content. High levels of total RNA, rRNA, and total and soluble protein were observed in treated S. acauke plants as compared with controls, but not in S. taberoswn. Levels of total lipid and phospholipid also were high in treated S. acauk plants as compared with controls but decreased in S. tuberosum during acclmation.When S. tuberosum, S. acaukl, and S. commersonii potatoes were coldtreated at constant day/night temperatures of 10, 5, and 2 C with 14-hour daylength, each species responds differently in terms of frost hardiness increase upon subjecting plants to a low temperature. For instance, after 20 days at 2 C, a net frost hardiness of 3 and 7 C was observed in S. acaude and S. coumersonii, respectively, whereas the frost hardiness in S. tuberoswn remained unchanged. Also, various levels of frost hardiness can be achieved in a species by subjecting plants to different low temperature treatments. Under a warm regime of 20/15 C day/night temperatures (14-hour light), both S. acauk and S. conunersonii can survive at -4.5 C or colder, whereas S. tuberosum can survive only at -2.5 C.Biochemical changes in the leaf tissue of these species were investigated at 5-day intervals during low temperature treatments. Increases in total sugar and starch were found in all three species during hardening, although S. tuberosum failed to harden. Soluble protein contents were increased in both S. acead and S. commersonii but decreased in S. tuberosunL RNA contents change in a pattern similar to the soluble protein. Net increases of the soluble proteins were positively and significantly correlated with net