1. methyl-(14)C-labelled 1,3-dihydroxy-4,5-dimethylbenzene, 5-methylorcylaldehyde and 5-methylorsellinic acid were synthesized from orcinol and sodium [(14)C]cyanide and tested for activity as precursors of gliorosein. ring-(14)C-labelled orcylaldehyde was also prepared. 5[(14)C]-Methylorcylaldehyde was incorporated into gliorosein (36% conversion); all the radioactivity was located in the C-methyl groups. 5-Methylorsellinic acid was decarboxylated by Gliocladium roseum and the resulting phenol was secreted into the medium. 2. The formation of an enzyme-bound derivative of 5-methylorsellinic acid as the first aromatic compound in the biosynthesis of gliorosein is suggested to explain these results. 3. ring-(14)C-labelled 3,4-dihydroxy-6-methyltoluquinone was also effectively incorporated into gliorosein and related products (20% conversion). 4. Sodium [(14)C]formate and [Me-(14)C]-methionine were incorporated into gliorosein and related products (15.4 and 22.2% conversion respectively). Isolation and estimation of the radioactivity in the O-methyl and C-methyl groups in the (14)C-labelled gliorosein thus formed showed an appreciable difference in the specific activities of the two types of methyl group (14 and 15% respectively). The results in the doubly-labelled methionine experiment indicate that the C-methyl group arises in the same manner as that in ergosterol; one of the original hydrogen atoms of the methyl group is lost. This confirms that C-methylation occurs at an ethylenic group at the aliphatic level. 5. The sequence of reactions at the aromatic level leading to the formation of gliorosein is proposed as 5-methylorsellinyl-enzyme-->3-hydroxy-5-methylorsellinyl-enzyme-->3,4-dihydroxy-6-methyltoluquinol-->3,4-dimethoxy-6-methyltoluquinol-->gliorosein.