The in situ perfused rat small intestine preparation was used to examine the extents of segmental absorption and metabolism of acetaminophen (A). Additionally, the preparation was employed to investigate any intestinal excretion of A and its conjugates from the circulation to the intestinal lumen. In this preparation, blood perfusate (300 ml) recirculated the intestinal preparation at 7.5 ml/min, entering via the superior mesenteric artery and returned to the reservoir via the portal vein. To demonstrate the extent of segmental absorption, metabolism, and excretion by different segments of the intestine, tracer doses of 3H-A (0.41 to 0.55 mumol in 0.3 ml of saline) were administered into the (a) entire intestine; (b) segments (first, second, and third) of one-third the length of the intestine, by instillation of the dose into the lumens of the segments; and (c) reservoir of perfusate. Exudates of luminal fluid from the injected segment and segments not exposed to drug were monitored for A and its conjugates during the experiment and at the end of 2 hr. Absorption of A was usually complete by 60 min; the extent of absorption of A at the end of 2 hr by the entire length of the intestine and by its three (first, second, and third) individual segments were 71.7 +/- 2.6, 50.5 +/- 4.0, 73.9 +/- 2.1, and 58.8 +/- 6.1% of dose (mean +/- SE), respectively. At the end of 2 hr, the total amount of acetaminophen glucuronide in perfusate and luminal fluid accounted for 3.1-5.5% and 0.14-0.1% of dose, respectively, among these preparations; acetaminophen sulfate was present only as a small percentage of dose in the lumen. Glucuronidation activity, when expressed as a percentage of the absorbed dose, was fairly constant for the entire intestine and first and second segments (8%) but decreased slightly for the third segment (7%). When A was present in blood perfusing the intestine, no metabolite was detected in perfusate or luminal fluid. Instead, unchanged A was excreted (5.6% dose) into the lumen. The effect of dose and vehicle on the extents of absorption and metabolism of A in the preparation was investigated by the instillation of different doses of A (0.16, 99.2, and 396.9 mumol in 0.3 ml of polyethene glycol 400) into the entire intestine at the duodenum.(ABSTRACT TRUNCATED AT 400 WORDS)