Actin stimulates the ATPase activity of myosin-S1. The regulatory proteins tropomyosin and troponin prevent this stimulation at conditions of low Ca2+ and low high-affinity myosin binding. At saturating Ca2+ and with strong actin-binding species of myosin (S1-ADP or NEM-S1) the enhancement of ATPase activity exceeds that seen in the absence of regulatory proteins. Inhibition and potentiation of actin activated ATPase activity are dependent on the position of tropomyosin on actin filaments. One position of tropomyosin (M state) leads to high activity whereas the other two positions (B and the intermediate C state) are inactive.We recently showed that the C-terminal 14 residues of troponin T are critical for formation of the inactive B state at low Ca2+. Furthermore, that same region of troponin T limits the formation of the M state at saturating Ca2+. Our present goal is to map the C-terminal region of troponin T to determine if specific residues in the sequence are responsible for each of these activities. The B state was monitored by changes in acrylodan tropomyosin fluorescence and by the rate of rigor S1 binding to regulated actin. The M state was monitored by an ATPase assay. Stepwise deletions within the C-terminal 14 amino acids of Troponin T progressively reduced the population of the inactive B state at low Ca2+. In contrast, the population of the M state was progressively increased at high Ca2+. The net positive charge of the last 14 residues appeared to be a key determinant of activity. Conclusion: The C-terminal region of troponin T plays essential roles in inactivation and in limiting activation. Both of these activities require basic amino acid residues within the COOH terminus.