Enrichment cultures yielded two strains of Pseudomonas putida capable of growth with orcinol (3,5-dihydroxytoluene) as the sole source of carbon. Experiments with cell suspensions and cell extracts indicate that orcinol is metabolized by hydroxylation of the benzene ring followed successively by ring cleavage and hydrolyses to give 2 mol of acetate and 1 mol of pyruvate per mol of orcinol as shown: orcinol leads to 2,3,5-trihydroxytoluene leads to 2,4,6-trioxoheptanoate leads to acetate + acetylpyruvate leads to acetate + pyruvate. Evidence for this pathway is based on: (i) high respiratory activities of orcinol-grown cells towards 2,3,5-trihydroxytoluene; (ii) transient accumulation of a quinone, probably 2-hydroxy-6-methyl-1,4-benzoquinone, during grouth with orcinol; (iii) formation of pyruvate and acetate from orcinol, 2,3,5-trihydroxytoluene, and acetylpyruvate catalyzed by extracts of orcinol, but not by succinate-grown cells; (iv) characterization of the product of oxidation of 3-methylcatechol (an analogue of 2,3,5-trihydroxytoluene) showing that oxygenative cleavage occurs between carbons bearing methyl and hydroxyl substituents; (v) transient appearance of a compound having spectral properties similar to those of acetylpyruvate during 2,3,5-trihydroxytoluene oxidation by extracts of orcinol-grown cells. Orcinol hydroxylase exhibits catalytic activity when resorcinol or m-cresol is substituted for orcinol; hydroxyquinol and 3-methylcatechol are substrates for the ring cleavage enzyme 2,3,5-trihydroxytoluene-1,2-oxygenase. The enzymes of this pathway are induced by growth with orcinol but not with glucose or succinate.