Pilocarpus microphyllus (Rutaceae), popularly known as jaborandi, is the only commercial source of an imidazole alkaloid named pilocarpine. In the present study, the variation in the profile of imidazole alkaloids in different seasons and in different parts of the P. microphyllus plant during the summer was analyzed by electrospray ionization mass spectrometry in the positive ion mode [ESI(+)-MS]. The fingerprints of these extracts repeatedly presented similar ions which were mass-selected and studied by tandem mass spectrometry (ESI-MS/MS and ESI-MS/MS/MS) and high-resolution mass spectrometry, resulting in the characterization of eight imidazole alkaloids. The data from the ESI(+)-MS fingerprints were analyzed by principal component analysis (PCA), showing that pilocarpine was present mainly in the summer, whereas in the autumn mainly pilosine and winter anhydropilosine were found. Three alkaloids, reported for the first time in extracts of P. microphyllus, were found. Analysis of the distribution of alkaloids in different parts of the plant during the summer showed that, although pilocarpine was present throughout the plant, 13-nor-8(11)-dihydropilocarpine was found mainly in the stem, pilosine and anhydropilosine were present mainly in the intermediary leaves, and the three new alkaloids were mainly found in the leaflets and petioles. Based on the dissociation patterns of these alkaloids, we observed that there were three structurally related groups of alkaloids differing in their distribution in the plant tissues and responding differently to seasonal variations. These results also indicate that these three groups of alkaloids could belong to intermediate, parallel or competitive pathways for pilocarpine formation biosynthesis. CI - Copyright (c) 2007 John Wiley & Sons, Ltd.