The grading of glial tumors has traditionally relied on histological assessment, but the distinction between grade II and grade III gliomas is still a subject of debate. We examined the value of the monoclonal antibody MIB-1 (Ki-67) labeling index (LI) in the differentiation between grade II and grade III gliomas by either the 1993 WHO grading scheme or the St. Anne-Mayo grading scale. The MIB-1 LI in the most densely labeled areas from 80 diffuse cerebral hemispheric gliomas was determined. The tumors included 16 grade II, 31 grade III and 33 grade IV gliomas by the WHO scale. The mean Lis (%) were 0.88 ± 0.29 for grade II, 8.752±1.71 for grade III, and 9.12 ± 1.55 for grade IV gliomas. Analysis of variance indicated a significant difference in mean LIs between grades II and III and grades II and IV (p ≤ 0.0001), but not between grades III and IV. Seven tumors were classified differently by the 2 systems (grade III by WHO, but grade 2 by St. Anne-Mayo), and all had MIB-1 LI over 3%. Univariate analysis showed that MIB-1 LI with a cut-off point at 1.5% was a significant prognostic factor (p ≤ 0.0005). High tumor grade (WHO, p ≤ 0.0002; St. Anne-Mayo, p ≤ 0.0006) and patient age >50 (p ≤ 0.0001) were also significant factors for shorter survival. Using Cox Regression Multivariate Analysis, MIB-1 LI>1.5% was a significant independent predictor of shorter disease survival when paired with tumor grade (p ≤ 0.032), patient age (p ≤ 0.0065), or gender (p ≤ 0.0007). We conclude that the MIB-1 immunoreactivity is useful in distinguishing grade II from grade III gliomas, and maybe more sensitive in assigning aggressive gliomas to grade III than the St. Anne-Mayo grading system.