The ionic character of antibiotics and suitable pH values for their extraction can simultaneously be determined by pH-chromatography. The authors utilized the analytical procedure in determining the ionic character of several antibiotics, followed by microbiological Rf determination. The following conclusions were made: (1) Antibiotics analyzed can be devided into four groups according to their ionic character, that is acidic, basic, neutral, and amphoteric compounds. (2) Penicillin and aureomycin are proved to be acidic, streptomyein to be basic, polymyxin-B to be neutral, and finally both chloromycetin and erythromycin to be amphoteric. The results on their ionic character with regard to their molecular structure were briefly discussed.