Abstract: Background. Aberrant promoter methylation of classical tumor suppressor genes occurs frequently during carcinogenesis. Several lines of evidences suggest that this epigenetic change also regulates microRNAs expression and may represent a potential molecular marker for cancer. Methods. We examined the methylation status at the hsa-miR-9-1 gene promoter in a series of 66 breast cancer cases by methylation sensitive PCR (MSP) analysis. For 43 of the 66 patients paired normal breast tissue and/or pre invasive (ADH, DCIS) lesions were also available. As control methylation status was determined on 6 normal breast tissues obtained from reductive mammoplasty.   Results. Methylation at mir-9-1 gene was detected in 32 out of 66 breast tumours (49%) and in none of the 6 normal breast tissues derived from reductive mammoplasty (P=0.02 χ2- Test). In all cases the same methylation status was demonstrated in tumour specimen, paired normal breast tissues and/or pre-invasive (ADH and DCIS) lesions. An higher frequency of methylation was found in patients showing metastases at diagnosis as compared with non metastatic patients (P=0.03 χ2-Test). Moreover, methylation at mir-9-1 gene was more frequent in patients showing bone metastases as first metastatic sites (P=0.04 χ2-Test), and in the subgroup of patients developing only bone metastases as compared with patients developing metastases  to visceral organs (P=0.03 χ2-Test). Conclusions. This study give further evidence of epigenetic mechanisms as regulators of miR-9 expression in breast cancer. Moreover, our results suggest an association between hypermethylation  at the miR-9-1 gene and metastatic site.