Compared with monolayer culture, tumour cells cultured as multicellular aggregates (spheroids) exhibit much higher levels of resistance to chemotherapeutic agents, a phenomenon known as multicellular resistance (MCR). Associated with multicellular aggregates is a heterogeneous microenvironment characterised by gradients in oxygen, pH, and nutrients. We previously showed that nitric oxide (NO) signalling plays an important role in the regulation of chemosensitivity in cancer cells cultured as monolayer, and that hypoxia increases resistance to anti-cancer agents largely through a mechanism involving the inhibition of NO signalling. The goal of the present study was to determine whether NO mimetics chemosensitize breast cancer cells in spheroid cultures. Survival of MDA-MB-231 breast carcinoma cells was determined by clonogenic assay following spheroid culture, doxorubicin exposure, and NO mimetic administration. When spheroids were incubated for 24 h with the NO mimetics diethylenetriamine/nitric oxide adduct (DETA/NO) and glyceryl trinitrate (GTN), cell survival after doxorubicin (200 microM) exposure was decreased by 33% (p<0.006) and by up to 47% (p<0.02), respectively. Nitric oxide-mediated signalling involves the generation of the second messenger cyclic guanosine monophosphate (cGMP). Administration of a non-hydrolysable cGMP analogue, 8-Bromo-cGMP, significantly decreased MCR (p<0.04). The effect of NO mimetic exposure on tumour cell chemosensitivity was not due to increased penetration of doxorubicin into spheroids, nor was it associated with an increase in cell proliferation. These results suggest that NO mimetics attenuate MCR to doxorubicin through a mechanism involving cGMP-dependent signalling. Therefore, NO-mimetics may potentially be used as chemosensitizers in cancer therapy.