In an attempt to improve liver slice culture for in vitro tests of newly developed drugs, we found that a relatively thick 2-mm slice was much more viable, long-lived, and metabolically functional than the conventionally used 300-μm-thick slices. Results revealed that some ionic and nonionic compounds (Na125I, [14C]Ala, and [14C]Xyl) dissolved in culture medium were absorbed into the slices, reaching from the cut edges deep into the inner part, in only a short period. Moreover, the slices could be observed for 24 h after incubation so that a normal histological image could be obtained. Each semi-microautoradiographic image was also clearly positive not only at the margin but also in the inner part of each slice. Furthermore, we determined the specific functioning of each donated liver for each substrate added, in uptake, metabolism, and excretion.