The morphine-induced increase in astrocyte and microglial immuno-labelling is caused by cell hypertrophy, not proliferation. Lumbar spinal cord sections were collected from animals administered BrdU (100 mg/kg) by intraperitoneal injection on days 1, 3, 5, and either intrathecal vehicle (saline; SAL) or morphine (15 μg; MS) once daily for five days by lumbar puncture. Representative photomicrographs acquired by confocal microscopy of spinal cord sections double labelled with 5-bromo-2-deoxyuridine (BrdU) and the astrocytic protein GFAP (B, C), the microglial marker Iba1 (E, F) or the neuronal marker MAP2 (H, I). No co-localization of BrdU-positive cells with GFAP or MAP-2-positive cells was observed. However, BrdU co-localized with a small number of Iba1 positive cells (arrow), suggesting a small portion of the newly formed cells were microglia or macrophages. (J). No difference was observed in the number of BrdU-positive cells in the dorsal horn (lamina II-IV) of lumbar spinal cord sections from animals administered chronic intrathecal saline or morphine. Data represent means ± s.e.m. for n = 6 sections per rat from n = 3 per group. Statistical analyses were performed by an un-paired t-test. ns = no significance. Scale bars, 30 μm.