To characterize the cellular density and proliferative activity of GFAP-negative cells in pilocytic astrocytoma (PA), surgically excised tissues of PAs (n=37) and diffuse astrocytomas (DAs) (n=11) were examined morphologically and immunohistochemically using antibodies against GFAP, Olig2, Iba1 and Ki-67 (MIB-1). In PA, Olig2 immunoreactivity was significantly expressed in protoplasmic astrocytes in microcystic, loose areas and cells in oligodendroglioma-like areas. Iba1-positive, activated microglia/macrophages were also commonly observed in microcystic areas. In compact areas, a prominent reaction for GFAP was observed, but for Olig2 and Iba1 to a lesser degree. On semiquantitative analysis, the number of Olig2-positive cells was significantly higher in PAs (mean labeling index (LI) +/- standard deviation (SD): 46.8+/-15.4%) than in DAs (13.3+/-7.8%) (P<0.001). Many Iba1-positive, microglia/macrophages were observed in PAs (19.9+/-6.5%), similarly to DAs (20.9+/-9.9%). Re-immunostaining of PA demonstrated that most Ki-67-positive, proliferating cells expressed Olig2, whereas GFAP or Iba1 expression in Ki-67-positive cells was less frequent (14.7+/-13.7%, and 8.8+/-13.6%) in a double immunostaining study. Conversely, the percentage of Olig2-positive, proliferating cells in total Olig2-positive cells (7.2+/-3.9%) was higher than that of Iba1-positive, proliferating cells in total Iba1-positive cells (0.9+/-0.6%). In conclusion, the present study found that PA consisted of numerous GFAP-negative cells, including Olig2-positive cells with high proliferation. Semiquantitative analysis of Olig2 immunohistochemistry in microcystic areas might therefore be useful for the differential diagnosis of PA and DA.