INTRODUCTION: Clinical resistance is a major factor limiting benefits to endocrine therapy. Causes of resistance may be diverse and the mechanism of resistance in individual breast cancers is usually unknown. The present study illustrates how changes in the expression of proliferation and oestrogen-regulated genes occurring during neoadjuvant treatment with the aromatase inhibitor, letrozole, may define distinctive tumour subgroups and suggest different mechanisms of resistance in clinically endocrine resistant breast cancers. METHODS: Postmenopausal women with large primary oestrogen-receptor (ER)-rich breast cancers were treated neoadjuvantly with letrozole (2.5 mg daily) for three months. Clinical response was determined by ultrasound changes in tumour volume. Tumour ribonucleic acid (RNA) from biopsies taken before, after 14 days and after three months of treatment was hybridized on Affymetrix U133A chips. Changes in expression of KIAA0101, TFF3, SERPINA3, IRS-1 and TFF1 were taken as markers of oestrogen regulation and those in CDC2, CKS-2, Cyclin B1, Thymidine Synthetase and PCNA as markers of proliferation. RESULTS: Fifteen tumours with < 50% volume reduction over three months of treatment were classified as being clinically non-responsive. Gene expression changes after 14 days of treatment with letrozole revealed different patterns of change in oestrogen regulated and proliferation genes in individual resistant tumours. Tumours could be separated into three different subgroups as follows: i) nine cases in which both proliferation and oestrogen signalling signatures were generally reduced on treatment (ii) four cases in which both signatures were generally unaffected or increased with treatment and (iii) two cases in which expression of the majority of oestrogen-regulated genes decreased whereas proliferation genes remained unchanged or increased. In 14 out of 15 tumours, RNA profiles were also available after three months of treatment. Patterns of change observed after 14 days were maintained or accentuated at three months in nine tumours but changes in patterns were apparent in the remaining five cancers. CONCLUSIONS: Different dynamic patterns of expression of oestrogen-regulated and proliferation genes were observed in tumours clinically resistant to neoadjuvant letrozole, thus illustrating heterogeneity of resistance and discriminating molecular sub-classes of resistant tumours. Molecular phenotyping might help to direct circumventing therapy suggesting the targeting of specific pathways in different tumour subtypes.