Isolated rat hepatocytes bind and internalize bovine lactoferrin (Lf) protein and Lf-bound Fe 3/ via Ca 2/ -dependent recycling Lf binding sites (McAbee, 1995, Biochem. J., 311:603-609). In this study, we determined if iron loading of primary cultures of adult rat hepatocytes altered their ability to bind and internalize Lf. Rat hepatocytes were cultured 16-24 h with or without ferric ammonium citrate (FAC) and then assayed for Ca 2/ -dependent 125 I-Lf binding at 4ЊC or 125 I-Lf endocytosis at 37ЊC. Cells pretreated with FAC (5 mg/mL) internalized two-to sixfold more 125 I-Lf than did control cells. The FAC-induced increase in 125 I-Lf endocytosis required 4-8 h of culture at 37ЊC and was fully reversible if cells were incubated an additional 24 h without FAC either in the presence or absence of the Fe 3/ chelator desferrioxamine. Maximal endocytic rates for untreated and FAC-treated cells were 370 and 2,300 molecules 125 I-Lf cell 01 sec 01 , respectively. Both 125 I-Lf binding at 4ЊC and endocytosis at 37ЊC increased up to sixfold between 0.3-10 mg/mL FAC, indicating that iron-induced enhancement of 125 I-Lf uptake was due to an increase in the number of Lf receptors present on the cells. 125 I-Lf bound to untreated and FAC-treated cells at 4ЊC with similar affinities (K d Ç 1.5 mM). Cycloheximide but not actinomycin D blocked the FAC-induced increase in 125 I-Lf binding, indicating that the increase in the number of Lf binding sites required translation but not transcription. Notably, iron loading blocked endocytosis of asialoorosomucoid by hepatocytes by up to 80%, reducing the number of active intracellular asialoglycoprotein receptors ú65% without altering the number of active cell surface receptors. We conclude from these studies that Lf receptor activity on hepatocytes is regulated posttranscriptionally by the iron status of the cells.