A trypsin inhibitor from leaves of field-grown alfalfa plants has been purified and shown to be the same trypsin inhibitor that is wound induced in leaves of young growth chamber grown plants. This inhibitor accounts for the major trypsin inhibitory activity found in both field-grown and wound-induced plants. The inhibitor exhibits a molecular weight of about 7500 and is specific for trypsin with a Ki of 1 X 10(-10) M. Analysis of the purified inhibitor by cation-exchange high-performance liquid chromatography revealed the presence of four isoinhibitor species that have identical immunological and inhibitory properties. The amino acid analysis of the four species indicates small but significant differences. Immunological double diffusion comparisons of the alfalfa inhibitor with the Bowman-Birk and Kunitz soybean inhibitors did not reveal any cross-reactivity although the amino acid content of the alfalfa inhibitor resembles those of Bowman-Birk family members.