Myopathy is the most common side effect of statins and its incidence is under-reported by randomised controlled clinical trials (1.5-5%) because of exclusion of susceptible individuals 1. The need to understand the mechanism of statin-induced myopathy is increasing as the cardiovascular risk threshold for statin prescription is reduced across the globe. We have previously shown that statin treatment increases the frequency and duration of Ca2+ sparks/embers in intact fast skeletal muscle fibres from the rat, consistent with increased sarcoplasmic reticulum (SR) Ca2+ leak (a common myopathic mechanism) 2. Here we investigate a role for nitric oxide (NO) and reactive oxygen species (ROS) in this increased leak. Male Wistar rats were treated with simvastatin 40 mg/kg/day by oral gavage over a 4 week period. Intact (non-permeabilised) type II skeletal muscle fibres from flexor digitorum brevis (FDB) were isolated by collagenase digestion and loaded with fluorescent dyes (Fluo-4AM, DAF-2 or JC-1). Data are given as mean ± S.E.M. of 17-50 cells from 5-11 rats, and compared with the Student’s t-test or 2 way ANOVA. Inhibition of NOS with L-NAME (1 mM) had a greater impact (P<0.05) on NO (indexed with DAF-2) in statin fibres compared with controls, consistent with increased NOS activity with statin treatment. Inhibition of NOS reduced spark frequency by 60% (P<0.01) in fibres from statin-treated animals, but was without effect in controls, suggesting a role for NO in increased leak. Expression of caveolin 1 and 3 (normalised to GAPDH) was reduced by 67% (P<0.05) and 24% (P>0.05) in gastrocnemius (GAS) muscle from statin treated animals; this could contribute to increased NOS activity as caveolins are the main constitutive inhibitors of nNOS and eNOS. There was also evidence of increased ROS production with statin treatment. Mitochondrial membrane potential (indexed with JC-1) was reduced in statin fibres compared with controls (1.1 ± 0.1 vs. 1.7 ± 0.2 AU; P<0.05). Uncoupling of the mitochondrial respiratory chain increases ROS production. We also saw a trend for a decrease in the ratio of reduced:oxidised gluthatione (GSH:GSSG) in GAS from statin-treated animals compared with controls (12 ± 1 vs. 14 ± 1; P>0.05). Importantly, the superoxide dismutase mimetic MnTMPyP (0.1 mM) and the mitochondrial ROS scavenger Mitotempo (25 µM) both significantly reduced (P<0.05) spark frequency in statin fibres but were without effect in controls. Together these data show that increased SR Ca2+ leak seen in intact muscle fibres from statin-treated rats is linked to an increase in NO and ROS. We propose that caveolin-regulated NO and Ca2+-dependent mitochondrial ROS production modify the RyR to effect this leak. Defining the cellular processes that underlie statin induced myopathy is the first step in the development of co-therapies to improve statin compliance.