Aims: To characterize and to purify a bacteriocin produced by Lactobacillus acidophilus strain with its activity restricted to Gram-positive bacteria. Methods and Results: Native acidocin CH5, a bacteriocin produced by L. acidophilus CH5 an isolate from a dairy starter culture forms in MRS (Oxoid, Basingstoke, UK) broth high-molecular weight aggregates which can dissociate into smaller units (retained by 5 kDa membrane) with higher activity. Acidocin CH5 was purified using combinations of chromatographic methods based on hydrophobic and cation exchange principles and the N-terminal region was sequenced. Conclusions: Based on Our results it is evident that acidocin CH5 belongs, according to bacteriocin classification, to the class II bacteriocins with identical N-terminal amino acid sequence described in the literature previously. Significance and Impact of the Study: The study has provided further data on bacteriocin acidocin CH5 from class II with wide spectrum of antimicrobial activity atypical for bacteriocins produced by L. acidophilus sharing the same homology.