Immunohistochemical staining is important for histological diagnosis of brain tumors; however, its intraoperative application has rarely been reported. Herein, we describe our methods and four successfully diagnosed cases. Between January 2008 and April 2010, intraoperative immunohistochemical analysis was performed in 43 patients undergoing brain tumor surgery at our institute. The time for rapid histological diagnosis was 70 min. MIB-1 immunostaining was performed; staining index (SI) was 0.8-76.2% (median, 2.5%) in rapid diagnoses and 0.6-83.9% (median, 7.7%) in permanent diagnoses. There was no discrepancy in low- or high-grade tumors between intraoperative and final pathological diagnosis. The antibodies used for staining were MIB-1 in all cases, L26 in 8 cases, UCHL-1 in 6 cases, GFAP in 4 cases, AFP in 3 cases, and PLAP in 5 cases. The staining patterns were similar between rapid and permanent diagnoses. We think that immunohistochemical examination is indicated under the following conditions: (1) preoperative radiologic differential diagnosis includes both high- and low-grade tumors, (2) intraoperative assessment is necessary to determine the extent of excision, and (3) quick and accurate pathological diagnosis is necessary for early initiation of treatment after surgery.