A major trypsin inhibitor, BTI-I, which had been prepared from broccoli (Brassica oleracea L.), inhibited bovine and porcine trypsins. The inhibition constants (K_i) for the BTI-I complexes with bovine and porcine trypsins were 6.4 × 10^-11 and 6.7 ×10^-11 M, respectively. The inhibitory specificity of BTI-I was examined with mammalian trypsins from the cat, horse, rabbit, sheep, dog, cow, guinea pig, rat, and goat. BTI-I inhibited each of these trypsins, although there were species differences. BTI-I was stable in the pH range of 2 to 8 during 24 h of incubation at 37 °C, and in acidic and neutral pH solutions after 30 min incubation at 100°C, although it was rapidly inactivated in alkaline solutions. The inhibitor was resistant to proteolytic digestion by bovine α-chymotrypsin, while it was relatively susceptible to the action of porcine pepsin.