Bac1829 fromStaphylococcus aureusKSI1829 is a newly identified peptide bacteriocin that inhibits a broad spectrum of bacteria. By taking advantage of its cationic and hydrophobic nature, a purification scheme was developed utilizing preparative isoelectric focusing and hydrophobic interaction chromatography. Due to a high pIvalue of approximately 9–10, 71% of the total Bac1829 activity was concentrated in two fractions by preparative isoelectric focusing. Final purification by high performance liquid chromatography on a propyl hydrophobic interaction column resulted in a 136-fold purification with an increase in specific activity from 181 to 24,623 antimicrobial units (AU)/mg protein. A total of 14,848 AU of Bac1829 were purified using this revised procedure, compared to 8702 AU as described previously. Mass spectrographic analysis of the purified sample yielded a single peak of 6418 ± 2 daltons. Since many bacteriocins possess similar biophysical attributes, the purification scheme presented in this communication may be applicable to those which have proven difficult to purify or have not been purified.