Eutherian mammal fast-twitch muscle fibres share similar contractile activation properties, suggesting that these properties are highly conserved in mammals. To investigate this hypothesis, we examined the contractile properties of skeletal muscle from the order Monotremata, a mammalian order that separated from eutherians 150 million years ago. The Ca²⁺- and Sr²⁺-activation properties of single mechanically skinned skeletal muscle fibres from the extensor digitorum longus (EDL) muscle of the short-nosed echidna were determined. Sigmoidal curves fitted to force response data plotted as a function of pCa (–log[Ca²⁺]), had a mean slope of 4.32 ± 0.28 and a mean pCa₅₀ and pCa₁₀ value of 6.18 ± 0.01 and 6.41 ± 0.02 respectively (n = 20). The mean pSr₅₀, pSr₁₀ and slope values of curves fitted to the force-response data after activation with Sr²⁺ were 4.80 ± 0.03, 5.29 ± 0.07 and 2.75 ± 0.18 respectively (n = 20). The mean pCa₅₀–pSr₅₀ value for the echidna EDL fibres was 1.37 ± 0.04. In five of the echidna fibres, exposure to submaximal Ca²⁺ concentrations produced myofibrillar force oscillations (mean frequency, 0.13 ± 0.01 Hz), a phenomenon found only in eutherian slow and intermediate muscle fibres. These results show that echidna EDL fibres generally have similar contractile properties to eutherian fast-twitch skeletal muscle fibres, such as those found in the EDL of the rat.