The metabolism of phenazopyridine was studied in the filamentous fungus Cunninghamella echinulata (synonym C. bainieri) ATCC 9244. Metabolic products were initially isolated by HPLC and TLC, with further characterization achieved by a combination of GLC, NMR, mass spectrum (MS), and GC–MS analyses. Selected samples of the incubation broths were treated with (β-glucuronidase–arylsulfatase. In addition to the reported mammalian metabolites p-aminophenol, acetaminophen, 2′-hydroxyphenazopyridine, 4′-hydroxyphenazopyridine, and parent drug, a novel metabolite was unequivocally identified as the sulfate monoester of 4′-hydroxyphenazopyridine.para-Hydroxylation of the aromatic ring with subsequent sulfoconjugation were observed as the major routes of metabolism. Ethanol, carbon monoxide, and quinidine had inhibitory effects on the C. echinulata metabolism of phenazopyridine, suppressing formation of 4′-hydroxyphenazopyridine and its sulfate monoester. Key words: Cunninghamella echinulata, biotransformation, GLC analysis of phenazopyridine, HPLC analysis of phenazopyridine, phenazopyridine, sulfate conjugate.