The effects of 4 weeks of thyroid hormone (3,5,3'-triiodothyronine, T3) treatment on the myosin isoform composition and maximum velocity of unloaded shortening (V0) of single soleus muscle fibres of young (3-6 months) and old (20-24 months) female (149 fibres) and male (200 fibres) rats were studied. Gender-related differences in the up-regulation of fast myosin heavy chain (MyHC) and myosin light chain (MyLC) isoforms were observed. In the female hyperthyroid rats, pure type I fibres and fibres co-expressing type I and type IIA MyHC (type I/IIA fibres) predominated. Some fibres expressed an alpha cardiac-like MyHC isoform either purely (alpha cardiac-like fibre type) or in co-expression with IIA MyHC (alpha cardiac-like/IIA fibre type). In the male hyperthyroid rats, on the other hand, all fibres were either type I/IIA or type I/IIAX. The relative quantities of fast MyLC isoforms in type I/IIA and type I/IIAX fibres was higher in female than in male hyperthyroid rats. V0 was similar in male and female control rats, and decreased with age in both genders (P<0.001). After T3 treatment, the average V0 increased (P<0.001) in females with a concomitant up-regulation of fast MyHC and fast MyLC isoforms irrespective of age. The average V0 of the pooled fibres was higher (P<0.001) in female than in male hyperthyroid rats at both ages. In conclusion, gender- and age-related differences were observed in the regulatory influence of 4 weeks' T3 treatment on myosin isoform composition and V0 in soleus fibres. These differences are presumably related to an interaction of thyroid and sex hormones in the regulation of myosin gene expression.