Compared with tumour cells cultured as sparse monolayers, tumour cells cultured as confluent monolayers exhibit high levels of resistance to anti-cancer agents. This phenomenon is called confluence-dependent resistance (CDR). We determined the contribution of hypoxia-inducible factor-1 (HIF-1), a key transcriptional regulator of cellular adaptations to hypoxia, to the development of CDR in MDA-MB-231 breast cancer cells. Clonogenic assays revealed a density-dependent increase in resistance to doxorubicin. Cell density also correlated with increased staining for reductively activated pimonidazole (a marker for hypoxia), as well as with increased levels of the HIF-1alpha subunit and HIF transcriptional activity as determined by immunocytochemistry, Western blot, and luciferase reporter assays. Importantly, inhibition of HIF-1alpha expression with siRNA significantly attenuated CDR. Similarly, shaking of cultures attenuated CDR, pimonidazole immunostaining, HIF-1alpha accumulation, and HIF-1 transcriptional activity. Having established a link between HIF-1 and CDR, we used HIF-1alpha and HIF-1 transcriptional activity as markers to investigate the role of additional factors in the regulation of CDR. Confluence-dependent increases in HIF-1alpha accumulation and HIF-1 transcriptional activity were observed even in cells cultured at 0.1% O(2) as well as in sparse cultures incubated with conditioned medium from confluent monolayers. Serum deprivation in both sparse and confluent cultures also resulted in HIF-1alpha accumulation. These results reveal that, although pericellular hypoxia may be a major contributor to HIF-1 activity, changes in the levels of soluble factors may also play a role. This study demonstrates that HIF-1 is required for CDR.