Presynaptic N-type Ca 2+ channels (Ca v 2.2, α 1B ) are thought to bind to SNARE (SNAP-25 receptor) complex proteins through a synaptic protein interaction (synprint) site on the intracellular loop between domains II and III of the α 1B subunit. Whether binding of syntaxin to the N-type Ca 2+ channels is required for coupling Ca 2+ ion influx to rapid exocytosis has been the subject of considerable investigation. In this study, we deleted the synprint site from a recombinant α 1B Ca 2+ channel subunit and transiently transfected either the wild-type α 1B or the synprint deletion mutant into mouse pheochromocytoma (MPC) cell line 9/3L, a cell line that has the machinery required for rapid stimulated exocytosis but lacks endogenous voltage-dependent Ca 2+ channels. Secretion was elicited by activation of exogenously transfected Ca 2+ channel subunits. The current-voltage relationship was similar for the wild-type and mutant α 1B -containing Ca 2+ channels. Although total Ca 2+ entry was slightly larger for the synprint deletion channel, compared with the wild-type channel, when Ca 2+ entry was normalized to cell size and limited to cells with similar Ca 2+ entry (≈150 × 10 6 Ca 2+ ions/pF cell size), total secretion and the rate of secretion, determined by capacitance measurements, were significantly reduced in cells expressing the synprint deletion mutant channels, compared with wild-type channels. Furthermore, the amount of endocytosis was significantly reduced in cells with the α 1B synprint deletion mutant, compared with the wild-type subunit. These results suggest that the synprint site is necessary for efficient coupling of Ca 2+ influx through α 1B -containing Ca 2+ channels to exocytosis.