Panaefluoroline B (2) is a fluorescent yellowish-green pigment produced by the cultured mycobiont of a lichen, Amygdalaria panaeola. Panaefluoroline B (2) has an isoquinoline skeleton, a C5 unit, and an amino acid, glycine, in its structure. The biosynthetic pathway of 2 was revealed by feeding experiments using [1-(13)C]-sodium acetate and [1,2-(13)C2][(15)N]-glycine. The analysis of labeling patterns of 2 and its mass spectrum suggested the isoquinoline part is biosynthesized via the acetate-malonate pathway with glycine as the nitrogen source and that the C5 unit originates from the mevalonate pathway.