Gap junctions are an important means for intercellular communication during development, processes of tissue differentiation, and in maintenance of adult tissue homeostasis. We investigated the expression levels and distribution of connexin‐43 (Cx‐43), the most abundant astrocytic gap junction protein, in acutely isolated astrocytes and glioma cells from biopsy tissue obtained from patients diagnosed with glioblastoma multiforme (GBM), low‐grade astrocytomas (LGAs), or mesial temporal lobe epilepsy. Western blot and immunohistochemical analyses indicated an inverse correlation between the amount of Cx‐43 protein and tumor malignancy grade, as assessed by calculating tissue mitotic indexes (MI) obtained using anti–Ki‐67 nuclear antigen staining. Samples from epilepsy patients had a low MI and were intensely positive for Cx‐43 staining, while LGA tissue samples exhibited moderate staining for Cx‐43 and average MI, and GBM biopsies showed significantly lower levels of Cx‐43 and high MI. Functional coupling was assayed using fluorescence recovery after photobleach (FRAP). We found that cells from glioma cell lines and primary cultures of human astrocytes from GBM tissues displayed significantly lower degrees of gap junction intercellular communication (GJIC) as indicated by longer and less complete recovery from photobleaching. Mean recovery values were GBM 23.8% ± 11.4%, LGA 49.4% ± 47%, and nontumor astrocytes 67.2% ± 8.4%. Western blot analysis of several human glioma cell lines and tissue biopsies showed variable expression levels of Cx‐43, which correlated negatively with the extent of recovery in the same samples. Taken together, our findings suggest that high‐grade brain tumors show reduced intercellular communication and a decrease in connexin‐43 protein levels. GLIA 33:107–117, 2001. © 2001 Wiley‐Liss, Inc.