Increase in citrate synthase (CS) activity in skeletal muscle is a well known adaptation to chronic aerobic exercise. However, skeletal muscle CS activity after a single bout of exercise is poorly understood. Some studies have shown no alteration (Cooper et al. 1986), increase (Tonkonogi et al. 1997; Leek et al. 2001) or even decrease (Ji et al. 1988) in CS activity in muscles extracted either from rodents or humans submitted to a single session of exercise. Here, we investigate the acute effects of muscle contractions on CS activity in rat skeletal muscle. We used male Albino rats (Wistar strain) weighing 250-275 g. The animals were anaesthetized by intraperitoneal injections of chloral hydrate (10% solution, 400 μl/100 g body wt). Subsequently, the hindlimb muscles were exposed and subjected to a protocol of electrical stimulation (n = 8). The protocol consisted of supra-maximal contractions (32 V) of 200 ms delivered every 1 s for 60 min (Wojtaszewski et al. 1997). The impulse frequency and duration within the contractions were of 100 Hz and 0.1 ms, respectively. One hindlimb side was stimulated to contract via the sciatic nerve (ipsilateral) while the opposite (contralateral) was maintained under resting conditions and used as a control. Rats were kept anaesthetized throughout the experiment by supplementing 200 μl of chloral hydrate every 30 min. After 1 h, both the stimulated and contralateral hindlimbs had their soleus muscles extracted for the determination of CS activity. We also had another group of non-stimulated anaesthetized rats (n = 7) that served as control. After the experiment, all animals were humanely killed. CS activity (mean ± S.E.M.) significantly increased by 62.2% in the stimulated soleus muscles (357 ± 24 nmol min^-1 mg^-1) and a surprising increase of 53.6% was also found in the contralateral non-stimulated soleus muscle (338 ± 24 nmol min^-1 mg^-1) compared to non-stimulated rats (220 ± 22 nmol min^-1 mg^-1). In order to elucidate if the contralateral increase in CS activity was caused by a neural mechanism, a second experimental group (n = 5) with the sciatic nerve sectioned only in the contralateral hindlimb was investigated. Interestingly, it was found that denervation abolished the ipsilateral-contraction-induced increase in CS activity in the contralateral limb. Taken together these data provide evidence for a novel mechanism involving neural regulation of CS activity in skeletal muscle.