Microglia are thought to play important roles not only in repairing injured tissue but in regulating neuronal activity, and visualizing the cells is very useful as a means of further investigating the function of microglia in vivo. We previously cloned the ionized calcium-binding adaptor molecule 1 (Iba1) gene, which is expressed selectively in microglia/microphages. To generate new transgenic mice to visualize microglia with enhanced green fluorescent protein (EGFP), we here constructed a plasmid carrying EGFP cDNA under control of the Iba1 promoter. This construct was injected into C57B/6 mouse zygotes, and the Iba1-EGFP transgenic line was developed. Fluorescent in-situ hybridization analysis revealed that the Iba1-EGFP transgene was located on chromosome 11D. No obvious defects were observed during development or in adulthood, and the EGFP fluorescence remained invariant over the course of at least four generations. Judging from the immunoreactivity with anti-Iba1 antibody, all EGFP-positive cells in the adult brain were ramified microglia. In the developing transgenic embryos, EGFP signals were detected as early as embryonic Day 10.5. The most prominent EGFP signals were found in forebrain, spinal cord, eye, foreleg, yolk sac, liver, and vessel walls. At postnatal Day 6, clear EGFP signals were observed in the supraventricular corpus callosum, known as “fountain of microglia,” where ameboid microglia migrate into the brain parenchyma and mature into ramified microglia. Iba1-EGFP transgenic mice thus permit observation of living microglia under a fluorescence microscope and provide a useful tool for studying the function of microglia in vivo. © 2005 Wiley-Liss, Inc.