Synaptotagmin 1 (Syt 1) functions as an essential Ca2+ sensor for the fast but not slow component of Ca2+-triggered exocytosis. One hypothesis to account for this selective function, based on the close homology of Syt 1 with synaptotagmin 9 (Syt 9), is that these Syts are redundant for the slow but not the fast component of release. We now show, however, that Syt 9 has unique properties that set it apart from Syt 1. Different from Syt 1, endogenous Syt 9 does not associate Ca2+ dependently or independently with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) protein complexes, and the Syt 9 C2B domain does not form Ca2+/phospholipid complexes, whereas such complexes are essential for Syt 1 function. Nevertheless, the C2A domain of Syt 9 functions as a Ca2+-binding module, suggesting that Syts 1 and 9 are Ca2+ sensors with similar Ca2+-binding sequences but distinct properties that indicate nonoverlapping functions.