There are two methods of assaying for naturally occurring α-amylase inhibitors. In the first method, (E + I) + S, where E is the enzyme, I is the inhibitor, and S is the substrate, the enzyme and inhibitor are preincubated for a period of time and residual activity is measured by adding substrate (starch). In the second method, (I + S) + E, the inhibitor and substrate are combined and the reaction is started by addition of enzyme. Although the first method can be used to measure the concentration of +-amylase inhibitors in a given plant or plant structure, it may give a somewhat biased view of their in vivo activity. The second assay method may be a much more realistic way to assess the effectiveness of the inhibitors in a feeding insect, because at least in granivorous insects that feed on wheat endosperm, ingestion of starch and the proteinaceous inhibitors probably occurs simultaneously. Consequently, assays were conducted by the second assay procedure with amounts of +-amylase found in guts of the rice weevil,Sitophilus oryzae(L.), and with amounts of +-amylase inhibitors present in wheat endosperm. With these conditions, inhibition of enzyme activity was reduced significantly compared with the nearly complete inhibition possible with preincubation. These findings indicate that in vitro methodology should simulate in vivo conditions as closely as possible to allow a more precise evaluation of the potential of naturally occurring inhibitors as resistance factors against insects.