AbstractIncubation of primary cultures of parenchymal hepatocytes in a conditioned medium (CM), collected over the first 3 h of serum‐free rat hepatocyte culture (CM0–3), induces a time dependent increase of the frequency of apoptotic cells which is accompanied by prominent changes of cell morphology. Short‐term treatment with CM0–3 for the first 3 h of culture is sufficient to significantly (P < 0.05) increase the frequency of apoptotic cells, however, the effect is more pronounced upon long‐term treatment. Although apoptosis induction by CM0–3 is independent of the timepoint when cultivation in CM0–3 starts, our results suggest that the sensitivity for apoptosis induction by CM0–3 is increased during the phase of attachment. Purification of CM0–3 resulted in a fraction which significantly (P < 0.05) induced apoptosis at concentrations ≥10 ng/ml. Exposure of cultures to concentrations ≥1 μg/ml of purified CM0–3 gave rise to a prominent cytotoxic effect as indicated by the massive occurrence of necrotic cells. Biochemical analysis showed that the purified fraction of CM0–3 contains acidic ferritins with molecular weight of 23 and 43 kDa. Strikingly, both share homologies with placental isoferritins (PLF), for which growth inhibitory and immunosuppressive effects have been demonstrated by several investigations. Therefore, our results provide evidence that rat hepatocytes produce PLF or PLF‐related acidic isoferritins which are able to induce apoptosis. J. Cell. Physiol. 198: 452–460, 2004© 2003 Wiley‐Liss, Inc.