Noninvasive examination of the hemodynamics of brain tissue is of general interest in many areas of medicine and physiology. To date, optical brain studies generate topographic maps, where signals obtained form within the brain are projected onto two-dimensional surface maps. Recently, our group has presented the first three-dimensional, volumetric reconstruction of hemodynamic changes during a Valsalva maneuver in the human forehead. To further validate our three-dimensional diffusion optical tomographic reconstruction algorithm we have turned to experimental studies involving small animals. Here we report on hypercapnia studies performed with 3-month old Sprague-Dawley rats. After anesthetizing the animal a tracheotomy was performed and the rat was artificially respirated. The head shaven and secured in a stereotaxic frame an optical probe was positioned between the bregma and lambda skull landmarks. A baseline measurement was recorded and then the inspired gas content was altered. The experimental studies verified the ability of our code to three-dimensionally visualize a global hemodynamic phenomenon in the rat head in response to perturbations in the inspired CO2 concentrations. Specifically, we incrementally increased the concentration of inspiratory CO2 (hypercapnia) and visualized the resulting hemodynamic change. We observed a global increase in blood volume and oxygenation, which was consistent with the known physiologic response to hypercapnia. A second set of experiments were designed to determine the sensitivity of the reconstruction to inaccurate probe positioning versus assumed model optrode-position mismatch. We determined that shifts on the order of 1/10 the maximum optrode separation significantly influence the reconstruction and may falsely produce lateralized effects.