The time-dependent effect of salicylic acid (SA) on alleviating cold damage in the Tokak (tolerant) and Akhisar (sensitive) varieties of barley (Hordeum vulgare) was investigated. Seedlings subjected to 0.0, 0.1, and 1 mM SA for 7 days were transferred to cold (7/5 degrees C) on days 7, 14, 21, and 28 for 3 days. It was found that 0.1 mM SA had a greater alleviating effect, decreasing the freezing injury in both varieties for at least 10 days after the application. Moreover, 0.1 mM SA had a greater effect on alleviation in Tokak by increasing the ice nucleation activity of apoplastic proteins extracted from the leaves. SA was effective for at least 24 days in decreasing the effect of freezing temperature by regulating apoplastic proteins in the leaves after the application. Furthermore, 0.1 and 1 mM SA had an alleviating effect by decreasing the lipid peroxidation level in both varieties for at least 24 days after the application. It can be seen that SA treatment can play a positive role in alleviating cold damage in barley. Therefore, it is suggested that results from SA treatments may contribute to research related to diminishing cold damage in agricultural applications.