Metformin treatment, now widely prescribed in PCOS, is aimed at correcting the associated insulin resistance, but it has also been shown to directly inhibit ovarian steroidogenesis. The mechanisms however, by which metformin inhibits oestradiol production in human granulosa cells remain unknown. Granulosa luteal cells were incubated with metformin, insulin or combined metformin and insulin treatment and aromatase mRNA expression was quantified using real-time PCR. Enzyme activity was assessed by the conversion of ³H-androstenedione to oestrone and oestradiol. Metformin's effect on the expression of specific untranslated first exon aromatase promoters was analyzed using semi-quantitative PCR. The involvement of MEK/ERK pathway was investigated by immunoblotting for aromatase, phosphorylated and total ERK-1,2 from cells cultured as above with/without the MEK inhibitor PD98059. Metformin significantly inhibited basal and insulin-stimulated aromatase mRNA expression, with parallel results from the aromatase activity assay and protein assessment. This suppression was via down-regulation of aromatase promoter II, I.3 and I.4 expression and was reversed by the addition of PD98059. Involvement of the ERK signalling pathway was demonstrated by the significant increase in phosphorylated ERK- 1,2 with the combined metformin and insulin treatement. We have shown for the first time in human granulosa cells that metformin significantly attenuated basal and insulinstimulated P450 aromatase mRNA expression and activity, via silencing of key promoters. This occurred by activation of MEK/ERK pathway which negatively regulates aromatase production. This is an important consideration given metformin's widespread use in PCOS and may further support a possible therapeutic indication in oestrogendependent breast tumours.