Biopsy samples were taken from vastus lateralis muscle of seven young (YO, age 30.2 ± 2.2 years), and seven elderly (EL, age 72.7 ± 2.3 years) subjects and two elderly subjects whose right leg had been immobilized for 3.5 months (EL‐IMM, ages 70 and 75). The following main parameters were studied: (1) myosin heavy chain (MHC) isoform distribution of the samples, determined by SDS‐PAGE; (2) cross‐sectional area (CSA), specific force (P_o/CSA) and maximum shortening velocity (V_o) of a large population (n= 593) of single skinned muscle fibres, classified on the basis of MHC isoform composition determined by SDS‐PAGE; (3) actin sliding velocity (V_f) on pure myosin isoforms determined by in vitro motility assays; (4) myosin concentration in single fibres determined by quantitative SDS‐PAGE. MHC isoform distribution was shifted towards fast isoforms in EL and to a larger extent in EL‐IMM. In EL and, more consistently, in EL‐IMM we observed a higher percentage of hybrid fibres than in YO, and noted the presence of MHC‐neonatal and of unusual hybrid fibres containing more than two MHC isoforms. P_o/CSA significantly decreased in type 1 and 2A fibres in the order YO → EL → EL‐IMM. V_o of type 1 and 2A fibres was significantly lower in EL and higher in EL‐IMM than in YO, i.e. immobilization more than counteracted the age‐dependent decrease in V_o. The latter phenomenon was not observed for V_f. V_f on myosin 1 was lower in both EL and EL‐IMM than in YO. V_f on myosin 2X was lower in EL than in YO, and a similar trend was observed for myosin 2A. Myosin concentration decreased in type 1 and 2A fibres in the order YO → EL → EL‐IMM and was linearly related to the P_o/CSA values of corresponding fibre types from the same subjects. The experiments suggest that (1) myosin concentration is a major determinant of the lower P_o/CSA of single fibres in ageing and especially following immobilization and (2) ageing is associated with lower V_o of single fibres due to changes in the properties of myosin itself, whereas immobilization is associated with higher V_o in the absence of a change in myosin function.