Inactivation of p16^INK4, an inhibitor of cyclin‐dependent kinases 4 (CDK4) and 6 (CDK6), may be essential for ontogenesis in non‐small cell lung cancer (NSCLC). We examined the sensitivity of two clones of P16^INK4‐transfected NSCLC cell line with homozygous deletion of p16^INK4, A549/pl6‐l and 2, to DNA topoisomerase I (topo I) inhibitors. A549/pl6‐l and ‐2 showed 7.7‐ and 9.1‐fold increases in sensitivity to CPT‐11 (11,7‐ethyl‐10‐[4‐(1‐piperidino)‐1‐piperidino]carbonyloxycamptothecin), respectively, compared with A549 cells. Ectopic p16^INK4‐expressing cells also showed ∼4.0‐fold increase in sensitivity to SN‐38 (7‐ethyl‐10‐hydroxycamptothecin), the active metabolite of CPT‐11, compared to the parent cells. The topo I‐mediated DNA relaxation activities of ectopic p16^INK4‐expressing cells were approximately 5 times higher than those of the parent cells. Northern and western blot analyses indicate that these increased topo I activities of ectopic p16^INK4‐expressing cells were due to an elevated topo I mRNA level and an increase in topo I protein. The chemosensitivity to topo I inhibitors, topo I mRNA level, protein content and activity of a pl6^INK4 revertant, lacking functional p16^INK4, tended to be restored toward those of the parental phenotype to some extent. These results suggest that p16^1NK4 expression is closely associated with the increased sensitivity of ectopic pl6^INK4‐expressing NSCLC cells to topo I inhibitors. The up‐regulation of topo I mRNA level, protein content and activity may he responsible for this hypersensitivity.