Jaborandi (Pilocarpus microphyllus) is the only known source of pilocarpine, and although this alkaloid is the only natural compound used to treat glaucoma, very little is known about its metabolism. Calluses obtained from petioles of P. microphyllus leaves were partially immersed in MS (Murashige and Skoog) liquid medium containing different pH levels (4.8, 5.8, and 6.8), nutrient concentration (MS normal basal medium concentration, absence of N, P, and K and three times normal concentrations), histidine and threonine (0.05, 0.15, and 0.75 mM), NaCl (25 and 75 mM) and polyethylene glycol (5 and 15%). Exposure to methyljasmonic acid (MJ) vapor was also investigated. The calluses were subjected to these conditions for 4 and 8 d under gentle agitation in the dark. Some calluses were also kept under continuous light. Pilocarpine was identified in the liquid medium by liquid chromatography–mass spectrometry/mass spectrometry. The alkaloid quantifications in the media and cells were carried out by high performance liquid chromatography (HPLC). The calluses maintained in the dark released the greatest quantities of pilocarpine into the medium. Methyljasmonate inhibited the release of pilocarpine in the medium. High pH (6.8), absence and excess of N, excess of P, and 0.75 mM of histidine and threonine induced the highest production of the alkaloid.