Cellular resistance to etoposide has been correlated both with reduced levels and an aberrant cytoplasmic accumulation of the drug`s target, topoisomerase IIα (topo IIα). It is not known, however, whether a cytoplasmic pool of topo IIα is sufficient to confer drug resistance on cultured mammalian cells. In our study, we have transfected mouse fibroblasts and human 293 cells with truncated forms of human topo IIα fused to GFP and have examined the transformants for the subcellular localization of topo IIα and their resistance to etoposide. Transient transfection resulted in high‐level expression of all GFP‐topo IIα fusions tested, whereas in stably transfected cells the levels varied significantly. Transfectants expressing a central or a carboxy‐terminal topo IIα domain (aa 428‐1504, 639‐1028 or 1028‐1504) accumulated high levels of the fusion proteins, while only very low amounts of GFP‐topo IIα proteins were observed in cell lines expressing constructs that retain the amino‐terminus of the enzyme (aa 1‐1214, aa 1‐939, aa 1‐611). Our results suggest that the topo IIα amino‐terminus affects the stability of truncated forms of the enzyme in mammalian cells, perhaps due to targeted degradation. Assays that screen for cell vitality and DNA synthesis reveal no significant changes in etoposide sensitivity in transfected cells expressing high levels of cytoplasmic or nuclear localized topo II fusion proteins. Retroviral expression of a cytoplasmically anchored domain of human topo IIα also failed to confer drug resistance. These results suggest that a cytoplasmic pool of topo IIα is not sufficient to render cultured mammalian cells drug resistant.