Synaptotagmin (syt) I is a Ca²⁺-binding protein that is well accepted as a major sensor for Ca²⁺-regulated release of transmitter. However, controversy remains as to whether syt I is the only protein that can function in this role, and whether the remaining syt family members also function as Ca²⁺ sensors. Elucidating the role of a specific protein such as syt I in Ca²⁺-dependent vesicle release may be facilitated if the protein could be eliminated from a model secretory cell system. In this study, we generated a PC12 cell line that continuously expresses a short hairpin RNA (shRNA) to silence expression of syt I by RNA interference (RNAi). Immunoblot and immunocytochemistry experiments demonstrate that expression of syt I is specifically silenced in cell lines that stably integrate the shRNA-syt I as compared to control cells stably transfected with the empty shRNA vector. The other predominantly expressed syt isoform, syt IX, was not affected nor was the expression of the SNARE proteins when syt I levels were knocked down. Syt I knockdown cells appear morphologically similar to control cells and are phenotypically differentiated with nerve growth factor. Resting Ca²⁺ and stimulated Ca²⁺ influx imaged with fura-2 were not altered in syt I knockdown cells. However, evoked release of catecholamine detected by carbon-fiber amperometry and HPLC was significantly reduced, but not abolished. Human syt I rescued the release events in the syt I knockdown cells. The reduction of stimulated catecholamine release in the syt I knockdown cells strongly suggests that although syt I is clearly involved in catecholamine release, it is not the only protein to regulate stimulated release in PC12 cells, and another protein likely has a role as a Ca²⁺ sensor for regulated release of transmitter.