In vitro root cultures of Anigozanthos preissii and Wachendorfia thyrsiflora (Haemodoraceae) are suitable biological systems for studying the biosynthesis of phenylphenalenones. Here we report how we used these root cultures to investigate precursor-product relationships between phenylpropanoids and phenylphenalenones whose phenyl rings share identical substitution patterns. Four phenylpropanoic acids, including ferulic acid and the unusual 4-methoxycinnamic acid, were used in (13)C-labeled form as substrates to study their incorporation into phenylphenalenones. In addition to the previously reported 2-hydroxy-9-(4'-hydroxy-3'-methoxyphenyl)-1H-phenalen-1-one (trivial name musanolone F), 2-hydroxy-9-(4'-methoxyphenyl)-1H-phenalen-1-one (proposed trivial name 4'-methoxyanigorufone) was found as a biosynthetic product in A. preissii. The carbon skeleton of 4'-methoxycinnamic acid was biosynthetically incorporated as an intact unit including its 4'-O-methyl substituent at the lateral phenyl ring. 4'-Methoxyanigorufone is reported here for the first time as a natural product.