The metabolic pathway of phenol was examined in the yeast strains of Rhodotorula (R.) rubra IFO 0892 and 1101. Changes in concentrations of phenol, phenol metabolites and dissolved organic carbon (DOC) in a medium were determined during incubation of phenol-grown cells with a phenol solution. A decrease in DOC concentration indicated that phenol was used as a carbon source. The phenol metabolites were separated and identified using high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). For HPLC analysis of β-ketoadipic acid, 2, 4-dinitrophenylhydrazone derivative was prepared. This analysis is very selective and sensitive. For GC/MS analysis, metabolites in the cultured broth were extracted with ethyl acetate and trimethylsilylated using N, o-bis(trimethylsilyl)acetoamide. Formation of muconolactone and β-ketoadipate enol-lactone from phenol was studied using whole cells. β-Ketoadipic acid was produced from muconolactone by the crude cell-free extract of R. rubra IFO 0892 and 1101. From the results in this study, a metabolic sequence for phenol degradation is proposed. Phenol may be hydroxylated to form catechol prior to ring cleavage, and catechol may be further oxidized to cis, cis-muconic acid, muconolactone, β-ketoadipate enol-lactone and β-ketoadipic acid. The catechol branch in the, β-ketoadipate pathway may exist in R. rubra and this catechol may be oxidized by the ortho type of ring fission.