Thrombin-induced platelet microbicidal protein (tPMP) is a small, cationic, antimicrobial peptide released from rabbit platelets when stimulated with thrombin. We studied the relationship between staphylococcal transmembrane potential (delta psi) and tPMP staphylocidal activity. A genetically related pair of Staphylococcus aureus strains, 6850 and JB1, which differ in delta psi generation (-143 and -97 mV, respectively) were used. Mutant JB-1 was substantially less susceptible to tPMP than the parental strain, 6850. Menadione supplementation, which normalized the delta psi of strain JB-1, did not restore JB-1 tPMP susceptibility. These findings suggest that the staphylocidal activities of tPMP require factors other than or in addition to an intact delta psi.