Pilocarpine, its stereoisomeride isopilocarpine, and pilocarpidine are the only thoroughly characterised alkaloids from different varieties of jaborandi leaves. Pilocarpidine, which does not contain an N-methyl group but possesses a free imino-group, gives a deep red coloration with Pauly's histidine reagent (sodium diazobenzene-p-sulphonate). The mother liquors from Pilocarpus microphyllus remaining after the separation of pilocarpine and isopilocarpine also exhibited this well-marked red coloration. A large quantity of such residues (representing many tons of leaves) was subjected to investigation to track the base containing the free imino-group using this colour reaction. Separation, mainly depending on the different solubilities of the bases in water, yielded: (1) resinous matter; (2) a sparingly soluble oil giving an intense Pauly reaction; (3) a sparingly soluble crystalline base designated pilosine (yielding 0.007 per cent. of the leaves, which did not give the Pauly reaction); (4) a mixture of pilocarpine and isopilocarpine; and (5) the mother liquor from (4) containing remaining readily soluble bases and giving only a moderate Pauly reaction. All attempts to isolate pilocarpidine from fraction 5 were unsuccessful, thus confirming Jowett's statement that P. microphyllus does not contain pilocarpidine. The base in fraction 2 (with Pauly reaction) was not isolated in crystalline form, but the new crystalline alkaloid pilosine from fraction 3 was characterised and investigated. Pilosine has the empirical formula C₁₆H₁₈O₃N₂ and is a monacid optically active base. It contains an N-methyl group but no methoxyl group, a lactonic grouping (accounting for two of the three oxygen atoms), and a hydroxyl group (the third oxygen atom). Treatment with acetic anhydride removes the elements of water to form a new unsaturated base, anhydropilosine (C₁₆H₁₆O₂N₂), which is a monacid optically active base with a lactonic grouping. When distilled with 20 per cent. aqueous potassium hydroxide, pilosine yields benzaldehyde and a new base pilosinine (C₉H₁₂O₂N₂), a monacid optically active crystalline base. Pilosinine is chemically, solubly, and physiologically similar to pilocarpine and isopilocarpine, and is highly probable to be a glyoxaline derivative and a lower homologue of pilocarpine and isopilocarpine. Physiologically, pilosine has a very weak pilocarpine action, anhydropilosine shows similar behaviour but is somewhat stronger, and pilosinine has a mild pilocarpine action.