Phosphorylation of oestrogen receptor alpha at serine 305 (ERalphaS305-P) induces tamoxifen resistance in experimental studies, but does not influence response to other endocrine agents, such as fulvestrant. We evaluated ERalphaS305-P using immunohistochemistry in 377 breast carcinomas from premenopausal participants of a randomized trial (n=248) and patients with advanced disease (n=129). Among the premenopausal patients, adjuvant tamoxifen improved recurrence-free survival (RFS) for ERalphaS305-P-negative tumours (multivariate HR=0.53, 95% CI 0.32-0.86, p=0.010), but not for ERalphaS305-P-positive tumours (multivariate HR=1.01, 95% CI 0.33-3.05, p=0.99) (interaction p=0.131). Notably, ERalphaS305-P was not significantly associated with RFS in patients not treated with tamoxifen (multivariate HR=0.64, 95% CI 0.30-1.37, p=0.248), indicating that ERalphaS305-P is a marker for treatment outcome rather than tumour progression. Given the direct experimental link between ERalphaS305-P and tamoxifen resistance and these first clinical data suggesting that premenopausal patients with ERalphaS305-P-positive breast cancer are resistant to adjuvant tamoxifen, further research is encouraged to study whether alternative endocrine treatment should be considered for this subgroup.