Cyclooxygenase-2 (COX-2) is associated with breast tumour progression. Clinical and molecular studies implicate human epidermal growth factor receptor 2 (HER2) in the regulation of COX-2 expression. Recent reports raise the possibility that HER2 could mediate these effects through direct transcriptional mechanisms. The relationship between HER2 and COX-2 was investigated in a cohort of breast cancer patients with or without endocrine treatment. A tissue microarray comprising tumours from 560 patients with 10-year follow-up was analysed for HER2, ERK1/2, polyoma enhancer activator 3 (PEA3) and COX-2 expression. Subcellular localisation of HER2 was assessed by immunofluorescence and confocal microscopy. Expression of markers examined was analysed in relation to classic clinicopathological parameters and disease-free survival in the presence and absence of tamoxifen. COX-2 expression associated with both membranous and nuclear expression of HER2 (P=0.0033 and P<0.00001 respectively). No association was detected between COX-2 and either ERK1/2 or PEA3 (P=0.7 and P=0.3 respectively). None of the markers were found to be independently prognostic. Membrane HER2, nuclear HER2 and COX-2, however, were all found to predict poor disease-free survival in patients on endocrine treatment (P=0.0017, P=0.0003 and P=0.0202 respectively). Moreover, patients who were positive for COX-2 predicted adverse effects of tamoxifen (P=0.0427). These clinical ex vivo data are consistent with molecular observations that HER2 can regulate COX-2 expression through direct transcriptional mechanisms. COX-2 expression correlates with disease progression on endocrine treatment. This study supports a role for COX-2 as a predictor of adverse effects of tamoxifen in breast cancer patients.