By means of the high sensitive on-line-coupled high-performance liquid chromatography and atmospheric-pressure chemical-ionization mass spectrometry (HPLC/APCI-MS and HPLC/APCI-MS/MS) techniques, the new macrocyclic spermine alkaloid prelandrine (5) was detected in the roots of Aphelandra squarrosa (Acanthaceae), and its structure was elucidated as 4'-hydroxyprotoverbine (=8-(4-hydroxyphenyl)-1,5,9,13-tetraazacycloheptadecan-6-one). It was further demonstrated that protoverbine (6) is enzymatically hydroxylated to prelandrine (5) in a reaction catalyzed by microsomes from the roots of A. squarrosa. The chemical synthesis of (-)-(S)-prelandrine is also described. The possible key role of prelandrine (5) as an intermediate in the biosynthesis of aphelandrine (1) is discussed.