It has been hypothesized that orotic acid (OA) promotes rat liver carcinogenesis by a differential mitoinhibitory mode. Consistent with this hypothesis, hepatic nodules are relatively resistant to OA-induced mitoinhibition. OA-induced mitoinhibition is dependent on the metabolism of OA to uridine nucleotides. The present studies investigate the uptake and metabolic pathway of OA, both in vivo and in vitro, as a possible basis for the resistance of hepatic nodules to OA-induced mitoinhibition. Rats bearing hepatic nodules exposed to 1% dietary OA exhibited increased levels of uridine nucleotides in the surrounding non-nodular liver (from 0.44 to 0.70 mg/g liver) but not in the hepatic nodules. Further, following administration of [3H]OA i.p., nodules have significantly lower levels of acid-soluble radioactivity compared to the non-nodular surrounding tissue. Furthermore, most of the acid-soluble radioactivity was present as uridine nucleotides, suggesting that the OA taken up was converted to uridine nucleotides. Similarly, hepatocytes from nodules in primary culture incubated with radiolabeled OA, have significantly lower levels (46-60%) of acid-soluble radioactivity. These results suggest that the decreased uptake of OA by hepatic nodules may be a factor contributing to the observed resistance of hepatic nodules to the mitoinhibitory effects of OA.