We investigated the expression and the functional properties of the slow skeletal troponin T (sTnT) isoforms in rat skeletal muscles. Four sTnT cDNAs were cloned from the slow soleus muscle. Three isoforms were found to be similar to sTnT1, sTnT2 and sTnT3 isoforms described in mouse muscles. A new rat isoform, with a molecular weight slightly higher than sTnT3, was discovered. This fourth isoform has never been previously detected in any skeletal muscle, and was therefore called sTnTx. From both expression pattern and functional measurements, it appears that sTnT isoforms can be separated in two classes, the high molecular weight isoforms (sTnT1 and sTnT2), and the low molecular weight isoforms (sTnTx and sTnT3). By comparing to the apparent migration pattern of the four recombinant sTnT isoforms, the newly described low molecular weight sTnTx isoform appeared predominantly and typically expressed in fast skeletal muscles, while the higher molecular weight isoforms were more abundant in the slow soleus muscle. The relative proportion of the different sTnT isoforms in the soleus was not modified after exposure to hindlimb unloading (HU), known to induce a functional atrophy and a slow-to-fast isoform transition of several myofibrillar proteins. Functional data gathered from replacement of endogenous troponin complexes in skinned muscle fibers show that the sTnT isoforms modified the calcium activation characteristics of single skeletal muscle fibers, with sTnT2 and sTnT1 conferring a similar increase in calcium affinity that was higher than that caused by the two low molecular weight isoforms sTnTx and sTnT3. Thus, we show for the first time the presence of sTnT in fast muscle fibers, and our data show that the changes in neuromuscular activity upon HU are insufficient to alter the sTnT expression pattern.