OBJECTIVES: To evaluate the specificity of three-color flow cytometry in childhood acute lymphoblastic leukemia (ALL) immunophenotyping. METHODS: Immunophenotyping was performed by three-color flow cytometry analysis using CD(45)/SSC gating. RESULTS: The percentage of blasts was correlated better with leukemic cell count compared with that of FSC/SSC, and the false positive results were low. Among eighty six cases of ALL, 95.3% was B-ALL, in which common-ALL and Pro-B-ALL were 76.8% and 6.1%, respectively, and 2.3% was T-ALL. CD(34)(+) and myeloid-associated antigen expression were observed in 57.0% and 34.9% of the cases, respectively, among which Pro-B-ALL was the commonest. CD(33) was more commonly expressed than CD(13) in Pro-B-ALL cases, but no difference in the expression between these two antigens in other subtypes. CONCLUSION: Gating of CD(45)/SSC eliminated effection of normal cells to blasts in bone marrow, with which the immunophenotyping results were more reliable.