The in vitro Ussing technique was employed to examine transport rates for acetaminophen and arginine across rabbit intestinal tissues. Mannitol and transepithelial conductance were used to monitor the integrity of rabbit intestinal tissues and the basal and stimulated short-circuit current were measured to assess functional viability. Transepithelial transport of acetaminophen, arginine, and mannitol was determined in rabbit jejunum, ileum, and distal colon. Transepithelial transport of arginine in the ileum and jejunum was composed of both passive (nonsaturable) (Pm = 0.06) and saturable components (KT = 0.6-0.7 m M; Jmax = 0.3-0.4 µmol/hr · cm2). The saturable component of arginine fluxes was abolished by pretreatment of the tissue with serosal ouabain (0.1 m M). In the distal colon, both unidirectional arginine fluxes were nonsaturable. In the segments examined, both unidirectional fluxes of acetaminophen were nonsaturable over the concentration range from 0.1 to 30 m M. These results provide values for maximal permeabilities attained by molecules traversing both the cellular and the paracellular pathways and, by comparison to their in vivo bioavailabilities, provide selection criteria for evaluating drug candidates for oral activity.