The absolute configuration of actinophyllic acid (-)-1, an alkaloid with an unprecedented 2,3,6,7,9,13c-hexahydro-1H-1,7,8-(methanetriyloxymethano)pyrrolo[1',2':1,2]azacino[4,3-b]indole-8(5H)-carboxylic acid skeleton isolated from Alstonia actinophylla, was determined through the study of its corresponding methyl ester 2. Racemic 2 was separated into (+)-2 and (-)-2 enantiomers, and they were assigned unambiguously as 15S,16R,19R,20R,21S and 15R,16S,19S,20S,21R, respectively, by the use of optical rotation and electronic circular dichroism. Finally, (-)-2 was characterized as the methyl ester of naturally occurring (-)-1. The assigned 15R,16S,19S,20S,21R-configuration of (-)-1 is consistent with a proposed biosynthetic pathway.