There are three different pathways by which phenacetin (P) can be converted to electrophilic reactive metabolites: 1) P is first deethylated to acetaminophen (A) which is subsequently activated by cytochrome P-450. 2) P is activated via an intermediate, possibly P 3,4-epoxide. 3) P is first converted to N-hydroxy-P, then activated by sulfation or glucuronidation. This work shows that these three pathways can be distinguised by metabolism of p-18O-P. 1) When p-18O-A was activated in vitro and in vivo there was a negligible loss of 18O in the A-GSH conjugate and the urinary A-mercapturic acid. 2) When p-18O-P was activated in vitro there was a 50 percent loss of 18O in the A-GSH conjugate. 3) When N-hydroxy-P-glucuronide was incubated with H218O there was a quantitative incorporation of 18O into the A-GSH conjugate. The relative in vivo importance of the three pathways was investigated by i.p. injection of 50 mg/kg of p-18O-P into hamsters. The urinary A-mercapturic acid showed approximately a 10 percent loss of 18O label indicating that pathway 1 is the predominant pathway.