Several in vitro and in situ approaches were used to determine the dominant presystemic activation site for (-)-6-aminocarbovir, (-)-carbocyclic 2',3'-didehydro-2', 3'-dideoxy-6-deoxy-6-aminoguanosine (6AC) conversion in rats. The in vitro disappearance half-lives (mean +/- S.D.) in the cytosolic fractions obtained from homogenates of the intestine, liver, and intestinal contents were 0.4 +/- 0.1 (n = 3), 12.2 +/- 1.1 (n = 3), and 15.5 (n = 1) min, respectively. An in situ vascularly perfused intestine-liver (IPIL) study was then carried out (n = 6) to determine the relative contribution of each presystemic organ to the overall first-pass extraction of 6AC. The 6AC extraction ratios in the intestine and liver in the IPIL were found to be 0.08 +/- 0.02 and 0.11 +/- 0.03, respectively. The intestinal extraction ratio was in dramatic contrast to the in vitro results. It was postulated that vascularly delivered 6AC had limited access to the metabolic site in the intestine. A theoretical analysis suggested that the extent of intestinal wall extraction of 6AC would be underestimated by the IPIL and should be determined after oral dosing. To compare intestinal extraction ratio in the IPIL with that after an oral administration, in situ intestinal lumen perfusions (n = 4) and intraportal infusions (n = 3) of 6AC were conducted in two groups of rats. The lumenally administered 6AC was extracted to a much greater extent by the intestine as compared with the IPIL, which presents 6AC to the intestine by the vascular route. The extraction ratio was found to be 0.54 +/- 0.06, which was significantly larger than that obtained in the IPIL.