Non-alcoholic fatty liver disease is the most common of all liver diseases. The hepatic disposition [³H]palmitate and its low molecular weight metabolites in perfused normal and steatotic rat liver was studied using the multiple indicator dilution technique and a physiologically based slow diffusion/bound pharmacokinetic model. The steatotic rat model was established by administration of 17-ethynylestradiol to female Wistar rats. Serum biochemistry markers and histology of treated and normal animals were assessed and indicated the presence of steatosis in the treatment group. The steatotic group showed a significantly higher ALT/AST ratio, lower levels of liver fatty acid binding protein and cytochrome P450 as well as microvesicular steatosis with an enlargement of sinusoidal space. Hepatic extraction for unchanged [³H]palmitate and production of low molecular weight metabolites were found to be significantly decreased in steatotic animals. Pharmacokinetic analysis suggested that the reduced extraction and sequestration for palmitate and its metabolites was mainly attributed to a reduction in liver fatty acid binding protein in steatosis.