Many xenobiotics are known to induce liver enlargement and hepatocarcinogenesis in rats, by mechanisms that remain largely undefined. As several of these compounds have significant human exposure, this has been cause for concern. The aim of this study, by investigating changes in gene expression occurring in conjunction with compound-induced increases in liver weight, was to identify molecular markers for the prediction of compound-induced hepatomegaly and hepatocarcinogenesis in vivo. The effects of four hepatomegalic compounds, namely phenobarbital, Wy-14,643, dexamethasone and cyproterone acetate were investigated in male Sprague-Dawley rats over a 72-hour time-course. The gene-hunting technique suppression subtractive hybridisation (SSH) isolated many potential differentially expressed gene transcripts from rat liver following Wy-14,643 treatment. Quantitative RT-PCR methods demonstrated four of these transcripts, namely carboxylesterase (Carb), cathepsin H (CatH), a Kupffer cell-surface receptor (KCR) and ubiquitin activating enzyme E1 (Ub), none of which have previously been reported to be involved in liver growth, to be differentially expressed following each of the compound treatments. KCR mRNA was demonstrated to be liver-specific, levels being down-regulated following treatment with three of the compounds. By analysis of liver sections, the cause of this down-regulation was attributed to decreased Kupffer cell numbers per field. Following dexamethasone treatment, this was correlated to substantial hepatocyte hypertrophy. However, the Wy-14,643 and cyproterone acetate-induced changes in Kupffer cell number are due to either a reduction in Kupffer cell number, or a change in their distribution due to the hyperplastic effects of these compounds. Kupffer cells are essential in the maintenance of liver homeostasis, capable of secreting various cytokines and growth factors, as well as antitumour agents, on activation. Recently, Kupffer cell-release of mitogenic cytokines was shown to be responsible for Wy-14,643-induced cell proliferation. A working hypothesis incorporating a novel role of Kupffer cells in xenobiotic-induced liver growth and hepatocarcinogenesis is therefore proposed.