The authors have developed a noninvasive optical method to image the subarachnoid space and cerebrospinal fluid pathways in vivo based on the near-infrared fluorescence of indocyanine green (ICG). The ICG was bound to purified lipoproteins (ICG-lipoprotein) and injected into the subarachnoid space of neonatal and adult rats. The ICG fluorescence was detected by a cooled charge-coupled device camera. After injection of ICG-lipoprotein into the cerebral subarachnoid space of the neonatal rat, ICG fluorescence was clearly detected at the injection site through the skull and skin. The ICG fluorescence was observed in the cerebellum and the lumbar spinal cord 1 and 8 hours postinjection, respectively. After injection of ICG-lipoprotein into the lumbar spinal subarachnoid space of an adult rat, ICG fluorescence was observed from the injection site to the thoracic levels along the spinal subarachnoid space. In addition, with the rat's head tilted downward, ICG fluorescence had extended to the cerebral subarachnoid space by 1 hour postinjection. The ICG fluorescence imaging of the cerebral subarachnoid space demonstrated an increase in fluorescence intensity around the lambdoid suture and the forebrain. On dissection of the rat brain the former location was identified as the supracerebellar cistern and the latter as the olfactory cistern. The results of this study are the first to demonstrate that an optical technique is applicable to imaging of the subarachnoid space and cerebrospinal fluid pathways in vivo. In addition, ICG-lipoprotein provides a sensitive optical tracer for imaging extravascular biological structures. Finally, ICG fluorescence imaging does not require an intricate imaging system because ICG is localized near the surface of the body.