Aims: To determine the factors affecting the release, stability and binding of bovicin HC5 to sensitive bacteria. Methods and Results: Stationary phase Streptococcus bovis HC5 cultures had little cell-free bovicin HC5 activity until the final pH was < 5.0, and even more bacteriocin was released by treatment with acidic NaCl (pH 2.0, 100 mmol l(-1)). Cultures grown with Tween 80 had more cell-free bovicin HC5 than untreated controls, but this nonionic detergent enhanced activity rather than release. Bovicin HC5 binding to S. bovis JB1 (a susceptible strain) was greater at pH values < 6.0. Bovicin HC5 bound other sensitive Gram-positive bacteria, but not Gram-negative species. Cultures retained most of their activity for 35 days, but only if the final pH was < 5.6. If the final pH was > 5.6, peptidases destroyed much of the activity. Conclusions: Bovicin HC5 remains cell associated until the culture pH is < 5.0, but it can be easily dissociated from the cell surface by acidic NaCl. It is highly stable in acidic environments and only binds sensitive bacteria at pH values < 6.0. Significance and Impact of the Study: Streptococcus bovis HC5 does not have generally regarded as safe status. However, bovicin HC5 has a broad spectrum of activity and sensitive bacteria do not become resistant. Based on these results, bovicin HC5 may be a useful bacteriocin model.