Technical Abstract: Bovicin HC5, a pore-forming lantibiotic produced by Streptococcus bovis HC5, has a broad spectrum of activity and can inhibit nisin-resistant bacteria. Experiments were designed to determine factors affecting the release, stability, binding and activity of bovicin HC5. Stationary phase S. bovis HC5 cultures grown with 2 mg glucose ml-1 (final pH 6.7) had little cell-free bovicin HC5 activity, but the specific activity (corrected for cell protein) increased when the glucose concentration was 10 mg ml-1 and the final pH was 4.7. When stationary phase S. bovis HC5 cells grown with 2 mg glucose ml-1 (final pH 6.7) were incubated in potassium phosphate buffer (10 mM) and the pH was decreased in stepwise fashion from 7.0 to 2.0, bovicin HC5 release was as much as 12-fold greater. Sodium chloride (100 mM) facilitated bovicin HC5 release at pH values greater than 2.0, but this effect was less than 2-fold. S. bovis HC5 cultures cultivated with Tween 80 (1 mg ml-1) had more cell-free bovicin HC5 activity, but subsequent work indicated this was a direct effect of Tween 80 on the activity of bovicin HC5 rather than its release from the cells. Acidic pH did not decrease the solubility of bovicin HC5 in basal medium. S. bovis JB1 (a susceptible strain) bound bovicin HC5, and this binding was much greater at acidic pH values. Bovicin HC5 bound other Gram-positive bacteria at pH 4.7 but it did not bind Gram-negative organisms or Gram-positives that have an outer membrane. S. bovis HC5 cultures retained virtually all of their activity for 35 days, but only if the glucose concentration was great enough to decrease the pH (< 5.6). If the final pH was > 5.6, activity could not be detected after 21 days.