A ffinity purification of crude acid extracts of rabbit polymorphonuclear leukocytes using Escherichia coli (J5) as adsorbent yields the bactericidal/permeability-increasing protein (BPI), two 15-kD species (p15s), and the two most potent (cationic) defensin species (neutrophil peptides [NP] -1 and -2). Tested in buffered isotonic medium, the relative antibacterial potency of these proteins against E. coli J5 is BPI (IC50 0.2 nM) > p15A (10 nM) > NP -1 (400 nM). Sublethal doses of p15A or NP-1 can synergize with BPI to decrease the dose required to inhibit the growth of E. coli by up to 50-fold. BPI and p15A display similar features of antibacterial action distinct from defensin NP-1, but NP-1 acts synergistically only with BPI and not with p15A. All aspects of the combined action of BPI and NP-1 resemble those observed with higher concentrations of BPI alone, implying that NP-1 enhances BPI potency. Neither NP-1 nor p15A alter the amount of BPI binding to E. coli but BPI enhances binding of p15A to E. coli, raising the possibility that synergy between these two proteins may occur at least partially at the level of binding. The potent synergistic actions of these proteins can also be demonstrated against serum-resistant clinical isolates of encapsulated E. coli tested in whole blood and plasma ex vivo, suggesting that such combined action may contribute to host defense in vivo.