Insulin resistance is a recognized feature of PCOS (polycystic ovary syndrome). However, the molecular reason(s) underlying this reduced cellular insulin sensitivity is not clear. The present study compares the major insulin signalling pathways in skeletal muscle isolated from PCOS and controls. We measured whole-body insulin sensitivity and insulin signalling in skeletal muscle biopsies taken before and after acute exposure to hyperinsulinaemia in nine women diagnosed with PCOS and seven controls. We examined the expression, basal activity and response to in vivo insulin stimulation of three signalling molecules within these human muscle samples, namely IRS-1 (insulin receptor substrate-1), PKB (protein kinase B) and ERK (extracellular-signal-regulated kinase) 1/2. There was no significant difference in the expression, basal activity or activation of IRS-1 or PKB between PCOS and control subjects. However, there was a severe attenuation of insulin stimulation of the ERK pathway in muscle from all but two of the women with PCOS (the two most obese), and an accompanying trend towards higher basal phosphorylation of ERK1/2 in PCOS. These results are striking in that the metabolic actions of insulin are widely believed to require the IRS-1/PKB pathway rather than ERK, and the former has been reported as defective in some previous PCOS studies. Most importantly, the molecular defect identified was independent of adiposity. The altered response of ERK to insulin in PCOS was the most obvious signalling defect associated with insulin resistance in muscle from these patients.