The effects of mustard trypsin inhibitor MTI-2 expressed at different levels in transgenic tobacco lines have been evaluated by feeding the lepidopteran Spodoptera littoralis throughout its larval life. Specific conditions were selected to study the long-term effects of feeding larvae on transgenic plants expressing the inhibitor at various levels. The data obtained led to the establishment of three relevant parameters to be considered during the experimentation: (i) the PI content of the plant lines to be used; (ii) the developmental stage of larvae sensitive to that PI content; (iii) the ratio of MTI-2/proteases sufficient to inhibit gut proteases. The experimental data obtained from feeding S. littoralis larvae using these conditions led to two main results. First, when L2 S. littoralis larvae were fed on high MTI-2 expressing tobacco plants, no effects on larval development were detected but there was a significantly reduced fertility. When the same larvae were fed on low expressing MTI-2 tobacco plants, only a less marked lowering of fertility was observed. Second, after the first generation, no differences in protease activity were observed in insects derived from larvae fed on high or low MTI-2 expressing tobacco lines, suggesting that genetic traits observed in previous studies were not inherited.