Reactive metabolites are considered to be responsible for toxic effects of phenacetin or p-phenetidine. The velocity of ferri-haemoglobin formation in dogs fed 13.7 mg (0.1 mmole)/kg p-phenetidine was increased by about 100% after phenobarbital (Phb) pretreatment (6×40 mg/kg). N-oxidation metabolites, estimated as the nitroso derivative, reached blood levels of 0.3 fig/ml in control dogs and about 1 μg/ml in phenobarbital pretreated dogs. Nitrosophenetol extracted into CCl4 from the blood of Phb treated dogs dosed with 0.5 mmoles/kg p-phenetidine was identified by thin layer chromatography, chemical reactions and UV-absorption. In dogs, the urinary excretion in 8 h of N-oxidation metabolites of p-phenetidine (160–280 μg, estimated in the form of p-nitrosophenetol) increased by 80–100% after Phb treatment. The half life in blood of intraveneously injected p-phenetidine was decreased from 90 to 40 min by Phb pretreatment. Rats given 0.5 mmoles/kg p-phenetidine orally, excreted in 10h, only about 0.1% of the dose in the form of N-oxidation products. The excretion of N-oxidation products in the urine of dogs fed 1 mmole/kg phenacetin was very low. In the urines of dogs pretreated with Phb 50–120 μg of N-oxidation products appeared during 8 h. Isolated microsomal fractions of rabbit liver and kidney catalysed the N-hydroxylation of p-phenetidine in vitro in the presence of O2 and NADPH2.10–20% of the substrate (1 μmole/ml) was N-hydroxylated in 10 min by liver microsomes of rabbits pretreated with Phb. Kidney microsomes of the same animals, on the other hand, N-hydroxylated only 3–6/o of the substrate in 10 min. N-hydroxylation products formed in the incubation mixtures were extracted in the form of p-nitrosophenetol into CCl4 after addition of 3mM K3[Fe(CN)6]. The nitroso compound was isolated in crystalline form and fully characterized. In vitro, the formation of ferri-haemoglobin in suspensions of bovine erythrocytes by p-nitrosophenetol is considerably increased by addition of washed microsomes + NADPH2. Therefore, the formation of ferri-haemoglobin in a system of microsomes, p-phenetidine, NADPH2 and erythrocytes cannot be used for a quantitative evaluation of oxidizing metabolites of p-phenetidine.