Cardiac troponin I (cTnI) is phosphorylated on three clusters of residues in response to protein kinase C (PKC) activation. Previously, studies on the cTnISer43/45 cluster showed phosphomimetic Asp substitution reduced peak shortening and accelerated re-lengthening in adult cardiac myocytes. The goal of the present study is to determine whether one or both Ser residues contribute to the functional response observed with cTnISer43/45Asp. We studied adult rat cardiac myocytes 2 and 4 days after viral-mediated gene transfer of cTnIFLAG, cTnISer43Asp or cTnISer45Asp (+FLAG). Western analysis indicated similar levels of cTnI replacement developed for all groups, and extensive replacement with cTnIFLAG (71±9%, n= 6), and FLAG-tagged epitopes of cTnIS43D (72±3%, n=8) and cTnIS45D (70±5%, n=8) within 4 days. Further analysis showed no significant change in cTnI stoichiometry and confocal analysis confirmed a sarcomeric incorporation pattern for each mutant. In functional studies, shortening amplitude decreased significantly in chronically paced myocytes expressing non-tagged Ser43Asp and/or Ser45Asp compared to controls (Control = 0.149±0.008 μm, n=36; cTnISer43/45Asp = 0.110±0.006 μm; n=32∗; cTnISer43Asp = 0.095±0.007, n=44∗; cTnISer45Asp = 0.108±0.007∗, n=50; ∗p<0.05 vs control) 4 days after gene transfer. An accelerated re-lengthening accompanied this reduced shortening (Time to 75% relaxation = TTR75% (ms): Control = 79±4; cTnISer43/45Asp = 62±4∗; cTnISer43Asp = 63±4∗, cTnISer45Asp = 65±3∗; ∗p<0.05 vs control). Interestingly, each single mutant also accelerated the time to peak shortening (TTP (ms): Control = 83±5; cTnISer43Asp = 68±3∗; cTnISer45Asp = 67±2∗; ∗p<0.05 vs control) while cTnISer43/45Asp did not (84±5). These initial results provide evidence that each Ser residue in the Ser43/45 cluster is capable of altering cTnI function in response to phosphorylation by PKC, yet phosphorylation of both residues does not produce an additive response.