Protease inhibitors (PIs) are plant compounds that can inhibit proteases of mammal, insect, or pathogen origin and are frequently induced by mechanical wounding, insect feeding, or pathogen infection. Nicotiana attenuata is a species that induces nicotine, volatiles, and phenolics in response to damage. Here we examine the distribution of PIs in N. attenuata to determine if they are part of the induced response in this species and if this response is ontogenetically constrained. We found that N. attenuata shoot extracts inhibited trypsin (Tryp) and chymotrypsin (Chym) activities, while root extracts inhibited Tryp, Chym, and the bacterial protease subtilisin (Sub). The highest TrypPI levels were found at midday in the source-sink transition leaf, while older or younger leaves contained lower TrypPI levels and did not show significant diurnal fluctuations. Rosette plants, bolting plants, and flowering plants all contained TrypPIs in leaves, stems, and flowers, while seed capsules, seeds, and young seedlings did not contain any PIs. PIs in N. attenuata rosette plants were induced by Manduca sexta larval feeding, methyl jasmonate (MeJA) treatment, wounding, and application of M. sexta oral secretion and regurgitant. The response to MeJA application was stronger and longer lasting than to mechanical wounding. The direction and magnitude of the systemic response to mechanical wounding or larval damage depended on the age of the leaf that was damaged and the frequency of wounding. The systemic signal for TrypPI induction appears to follow source-sink relations in the plant and to be regulated by the octadecanoid pathway. Interestingly, by the time plants reach the flowering stage, they had lost the ability to increase PI levels after MeJA treatment. We concluded that plant ontogeny constrains both constitutive and inducible PI production in N. attenuata.