Antifreeze proteins (AFPs) accumulate in the leaves of barley during cold acclimation, where they may inhibit ice recrystallization and produce freezing resistance of the plant. Four parental diallel crosses of the barley varieties were used to determine the heritability of AFPs and the relationship between the accumulation level of AFPs and freezing resistance. The concentration of apoplastic proteins in the cold-acclimated leaves was increased in the mean by four-fold over as compared with that of nonacclimated. The diallel cross analyses revealed that the gene of Sacheon 6 was dominant and those of Reno and Dongbori 1 were recessive. The AFPs had high narrow-sense heritabilities. The general combining ability effects of Reno and Dongbori 1 were much higher than the other parents. The bands of 32-kD for GLP, 35-& 28-kD for CLP and 25-, 22- & 16-kD for TLP were observed in the apoplastic extracts from cold-acclimated plants, but there were no clear differences between the parents and Fl hybrids. The concentrations of AFPs were significantly correlated with the degree of freezing resistance, indicating that the concentration of AFPs in the plant is the very important factor for freezing resistance.