The effect of acetylsalicylic acid (ASA) on the toxicity and metabolism of [¹⁴C]acetaminophen was studied in the mouse. Pretreatment with ASA did not affect lethality, but hepatotoxicity as determined by plasma transaminases was reduced by ASA pretreatment. The blood concentration profiles of radioactivity were altered by ASA following po and ip administration. It was suggested that ASA reduced the rate of acetaminophen absorption and inhibited elimination. Paper chromatography of urine showed that following ip dosing ASA reduced the excretion of sulfate conjugate but increased the excretion of total catabolites of the glutathione conjugate. In the po study a similar inhibition of sulfation was observed, but excretion of total glutathione degradation products was not altered statistically even though excretion of mercapturate was statistically elevated by ASA pretreatment. An attempt was made to correlate the excretion of glutathione degradation products (an estimate of the toxic pathway) with toxicity. A direct correlation could not always be demonstrated, and it was concluded that factors additional to toxic metabolite formation modified acetaminophen-induced hepatotoxicity in the mouse.