Biopsy samples were taken from vastus lateralis muscle of 7 young (YO, age 30.2±2.2 years), 7 elderly (EL, age 72.7±2.3 years) and 2 elderly subjects whose right leg had been immobilized for 3.5 months (EL- IMM, age 70 and 75). The following main parameters were studied: myosin heavy chain (MHC) isoform distribution of the samples by SDS-PAGE; cross sectional area (CSA), specific force (Po/CSA), maximum shortening velocity (Vo) of a large population (n=593) of single skinned muscle fibres, classified on the basis of MHC isoform composition by SDS-PAGE; actin sliding velocity (Vf) on pure myosin isoforms by in vitro motility assays; myosin concentration in single fibres 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 a higher percentage of hybrid fibres than in YO, the presence of MHC- neonatal and of unusual hybrid fibres containing more than two MHC isoforms were observed. Po/CSA significantly decreased in type 1 and 2A fibres in the order YO EL EL-IMM. Vo 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 Vo. The latter phenomenon was not observed for Vf. Vf of myosin 1 was lower in both EL and EL-IMM than in YO. Vf of myosin 2X was lower in EL than in YO, and a similar trend was observed for 2A myosin. Myosin concentration decreased in type 1 and 2A fibres in the order YO EL EL-IMM and was linearly related to the Po/CSA values of corresponding fibre types from the same subjects. The experiments suggest that (i) myosin concentration is a major determinant of the lower Po/CSA of single fibres in ageing and especially following immobilization and (ii) ageing is associated with lower Vo of single fibres due to changes in the properties of myosin itself, whereas immobilization is associated with higher Vo in the absence of a change in myosin function.