A barley cDNA clone encoding a cysteine proteinase inhibitor was characterized. The deduced amino acid sequence of this barley cystatin (Hv-CPI) contains the motif QXVXG conserved among members of the cystatin superfamily. The gene (Icy), located on chromosome 2, was expressed in embryos, developing endosperms, leaves and roots as assessed by northern blot analysis. Western blot analysis detected a slightly retarded band in leaves that was not present in roots or seeds. In these two organs a more precise location of Hv-CPI was done by immuno-histochemical analysis, with polyclonal antibodies raised against the recombinant CPI protein expressed in Escherichia coli. This protein efficiently inhibited papain (Ki 2.0 x 10(-8) M) and ficin (Ki 2.2 x 10(-8) M) and, to a lesser extent, chymopapain (Ki 1.6 x 10(-7) M) and was inactive against bromelain. The Icy mRNA expression in vegetative tissues increased in response to anaerobiosis, dark and cold shock (6 degrees C).