A paucity of information exists concerning the functional roles of the human slow skeletal troponin T isoforms (HSSTnT isoforms) in different muscle types. Three HSSTnT isoforms have been found in slow skeletal muscle: HSSTnT1 (+ Exons 5 and 12), HSSTnT2 (+5, −12), HSSTnT3 (−5, −12) and HSSTnT4 (−5, +12, only found at the mRNA level). Soleus rabbit skinned fibers were displaced with HSSTnT1, 2, 3 or 4 and reconstituted with human SSTnI-C/STnC complex. The extent of Tn displacement was analyzed by measuring the Ca2+ unregulated force (UF) at pCa 8.0 after SSTnT treatment. The UF ranged from 63 to 73%. The Ca2+ sensitivity increased between SSTnT isoforms: isoform 1 (pCa50 = 5.73) < isoform 2 (pCa50 = 5.80) < isoform 3 (pCa50 = 5.84). HSSTnT4 yielded a pCa50 = 5.78. Using a reconstituted fast skeletal muscle system, the actomyosin ATPase activity containing different HSSTnT isoforms was determined. The HSSTnT isoforms did not alter ATPase activation or inhibition in the presence or absence of Ca2+. Potential interactions between human cardiac troponin C (HcTnC), rabbit skeletal tropomyosin (RsTm) and human cardiac troponin I (HcTnI) with SSTnT were mapped. Dot blot analysis using HRP conjugated proteins revealed new interactions between SSTnT peptides and HcTnC, RsTm and HcTnI. These results may help identify the functional differences that occur between SSTnT isoforms due to their alternative splicing. Supported by NIH HL-042325(JDP) and AR-050199 (JDP) and AHA 0825368E (JRP).