To examine the expression patterns of myosin heavy chain (MHC) isoforms in single fibres of the soleus muscle following weightlessness, 10-week-old male Wistar rats were subjected to hindlimb suspension for 4 weeks. Hindlimb suspension resulted in reduced body weight and absolute and relative mass of the soleus muscle compared with controls (P < 0.01). A total of 975, 892 and 1098 single fibres from pre-suspended controls, age-matched controls and suspension groups, respectively, were subjected to MHC analyses using SDS-PAGE. Single fibres containing only MHC I decreased (87.9 vs. 67.9%, P < 0.05) and single fibres containing only MHC IIa disappeared after hindlimb suspension. On the contrary, single fibres containing multiple type II MHC isoforms were observed as follows: 10.1% single fibres contained MHCs IIa and IId; 14.1% contained MHCs I, IIa and IId; and some (1.4%) expressed the MHC IIb isoform with MHCs IIa and IId. The relative content (%) of each MHC isoform in MHC hybrid single fibres was calculated using densitometer scanning. The MHCs IIa and IId hybrid single fibres contained the same amount of MHC IIa (51.3 +/- 6.3%) and MHC IId (48.7 +/- 6.3%). In the MHCs I, IIa and IId hybrid single fibres, the percentage of MHC IIa was distributed in a wide range (approximately 80%), whereas the percentage of MHC IId was a relatively low range (approximately 40%), and the relative content of MHC I was inversely correlated with that of MHC IIa and MHC IId, respectively. The fibre type composition of suspended soleus muscle, analysed by histochemical myosin ATPase staining, was changed, with a decrease in the percentage of type I fibres and an increase in that of type IIA fibres. Our results indicate that hindlimb suspension induces multiple type II MHC expression in the soleus single fibres and suggest that the single fibres containing multiple type II MHC isoforms should be classified into type IIA.