Phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P₂) synthesis is required for calcium-dependent exocytosis in neurosecretory cells. We have developed a PtdIns(4,5)P₂ bead pulldown strategy combined with sub-cellular fractionation to identify endogenous chromaffin granule proteins that interact with PtdIns(4,5)P₂. We have identified two synaptotagmin isoforms, synaptotagmins 1 and 7, spectrin, alpha-adaptin and synaptotagmin-like protein 4 (granuphilin) by mass spectrometry and Western blotting. This pulldown strategy has also been used in combination with other methods to characterize the mechanisms of interaction of synaptotagmin 7 with PtdIns(4,5)P₂. The 45 kDa isoform of synaptotagmin 7 was found to be highly expressed in adrenal chromaffin cells compared to PC12 cells and to mainly localise to secretory granules by sub-cellular fractionation, immunoisolation and immunocytochemistry. We have demonstrated that synaptotagmin 7 binds PtdIns(4,5)P₂ via the C2B domain in the absence of calcium and via both the C2A and C2B domains in the presence of calcium. We have mutated the poly-lysine stretch in synaptotagmin 7 C2B and have demonstrated that this mutant domain lacks the calcium-independent PtdIns(4,5)P₂ binding. Synaptotagmin 7 C2B domain inhibits catecholamine release from digitonin-permeabilised chromaffin cells and this inhibition is abrogated with the C2B poly-lysine mutant. These data indicate that synaptotagmin 7 C2B–effector interactions which occur via the poly-lysine stretch, including calcium-independent PtdIns(4,5)P₂ binding, are important for chromaffin granule exocytosis.