The cytostatic drug bleomycin (BLM) induces pulmonary fibrosis as its main side effect. Fibroblasts in fibrotic foci are the main cellular source for extracellular matrix accumulation that typifies fibrosis. In vitro studies demonstrated the ability of cytotoxic drugs to induce terminal differentiation of fibroblasts. These postmitotic cells are very active in regard to production of collagens. The present study was addressed to investigate the potential of BLM to induce terminal differentiation of rat lung fibroblasts in vitro and the consequences for collagen production and for the expression and activity of the collagen modifying enzyme prolyl 4-hydroxylase (P4H). The BLM effects were compared with those of mitomycin C (MMC), another cytotoxic agent with known potential for initiation of postmitotic differentiation of fibrobasts. BLM induced postmitotic differentiation of rat lung fibroblasts. The capacity of the cells to form clones was diminished by BLM or MMC in a concentration dependent manner. Both drugs initiated the formation of an increasing number of postmitotic cell clones. The postmitotic differentiation was accompanied by an increase in total collagen production by the cells. Administration of BLM to cultures of lung fibroblasts at concentrations of 1 or 10 mU/ml resulted in an increase of the collagen amount to about the 1.5-fold and 1.6-fold of controls, respectively. Treatment of fibroblasts with MMC elevated the collagen level to about the 2-fold. P4H activity and P4Hα mRNA levels in cells exposed to BLM or MMC were found to be increased. We conclude that terminally differentiated fibroblasts might be part of the heterogeneous population of fibroblast-like cells in fibrotic foci responsible for the increased production of collagen during the fibrotic phase of the development of pulmonary fibrosis.