The LE IIIb fraction of the bacteriolytic enzyme from Staphylococcus hyicus could be isolated by ionic exchange chromatography and subsequent gel filtration. Isoelectric focusing of the highly purified enzyme preparation revealed an isoelectric point at pH 10.3. The lytic activity of LE IIIb on streptococci of various serogroups could be effectively analyzed with the help of an aggregometer and expressed as increase of transmittance at 546 nm. The streptococci differed in their LE IIIb lysis pattern, which was more pronounced with those of serological groups C and D and Streptococcus uberis. The lytic effect of the LE IIIb fraction was optimal at low molarity (0.01 mol/l) of the lysis buffer and reduced in the presence of HgCl2 and EDTA.