Membrane fusion was studied using human neutrophil plasma membrane preparations and phospholipid vesicles approximately 0.15 μm in diameter and composed of phosphatidylserine and phosphatidylethanolamine in a ratio of 1 to 3. Liposomes were labeled with N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD) and lissamine rhodamine B derivatives of phospholipids. Apparent fusion was detected as an increase in fluorescence of the resonance energy transfer donor, NBD, after dilution of the probes into unlabeled membranes. 0.5 mM Ca²⁺ alone was sufficient to cause substantial fusion of liposomes with a plasma membrane preparation but not with other liposomes. Both annexin I and des(1–9)annexin I caused a substantial increase in the rate of fusion under these conditions while annexin V inhibited fusion. Fusion mediated by des(1–9)annexin I was observed at Ca²⁺ concentrations as low as aproximately 5 μM, suggesting that the truncated form of this protein may be active at physiologically low Ca²⁺ concentrations. Trypsin treated plasma membranes were incapable of fusion with liposomes, suggesting that plasma membrane proteins may mediate fusion. Liposomes did not fuse with whole cells at any Ca²⁺ concentration, indicating that the cytoplasmic side of the membrane is involved. These results suggest that annexin I and unidentified plasma membrane proteins may play a role in Ca²⁺-dependent degranulation of human neutrophils.