The skeletal muscle exhibits large functional differences depending on the myosin heavy chain (MHC) isoform expressed in its molecular motor myosin II. The differences in the mechanical features of force generation by myosin isoforms are investigated in situ by using fast sarcomere-level mechanical methods in permeabilised fibres (sarcomere length 2.4 μm, temperature 12°C, 4% dextran T-500) from the slow (soleus, containing the MHC-1 isoform) and the fast (psoas, containing the MHC-2X isoform) skeletal muscle of the rabbit. The stiffness of the half-sarcomere was determined at the plateau of Ca2+ -activated isometric contractions and in rigor and analysed with a model that accounts for the filament compliance to estimate the stiffness of the myosin motor (ε). ε is 0.56 ± 0.04 pN nm-1 and 1.70 ± 0.37 pN nm-1 for the slow and fast isoform respectively, while the average strain per attached motor (s0 ) is similar (≈ 3.3 nm) in both isoforms. Consequently the force per motor (F0 = ε ⋅ s0 ) is three times smaller in the slow isoform than in the fast isoform (1.89 ± 0.43 pN versus 5.35 ± 1.51 pN). The fraction of actin-attached motors responsible for maximum isometric force at saturating Ca2+ (T0,4.5 ) is 0.47 ± 0.09 in soleus fibres, 70% larger than that in psoas fibres (0.29 ± 0.08), so that F0 in slow fibres is decreased by only 53%. The lower stiffness and force of the slow myosin isoform open the question of the molecular basis of the higher efficiency of slow muscle with respect to fast muscle. This article is protected by copyright. All rights reserved.