1. Single skeletal muscle fibres from a mouse foot muscle were used to investigate the effects of nitric oxide on contractile function. 2. We measured force production and myoplasmic free Ca2+ concentration ([Ca2+]i) in single fibres exposed to the nitric oxide donors S-nitroso-N-acetylcysteine (SNAC) and nitroprusside. 3. The nitric oxide donors reduced myofibrillar Ca2+ sensitivity, whereas [Ca2+]i transients were increased during submaximal tetani. Force was largely unchanged. SNAC did not change maximum shortening velocity, the rate of force redevelopment, or force production at saturating [Ca2+]i. 4. The guanylyl cyclase inhibitor LY83583 increased tetanic [Ca2+]i but had no effect on Ca2+ sensitivity. LY83583 did not prevent the decrease in myofibrillar Ca2+ sensitivity in response to SNAC. The oxidizer sodium nitrite increased tetanic [Ca2+]i and decreased myofibrillar Ca2+ sensitivity. 5. We conclude that under our experimental conditions nitric oxide impairs Ca2+ activation of the actin filaments which results in decreased myofibrillar Ca2+ sensitivity.