A technique employing diffuse reflectance spectroscopy (DRS) is described to assess and mirror dynamic changes of pancreatic tissue perfusion. An especially designed reflectance spectrophotometer was initially used to derive the quantitative relation between hemoglobin concentration ([Hb]) and reflectance measurements in vitro. Over a wide range of scattering related to the medium in which the measurements were made (scattering coefficient: 6.5-13 cm-1), a close, direct correlation existed with a slope of 0.376 +/- 0.012. In Sprague-Dawley rats under general anesthesia, the pancreas was isolated in situ and perfused with graded infusions of hemoglobin solutions. A correlation, comparable to the in vitro setting, was found between a [Hb] of 0 and 14 g/dl in the perfusate with slopes of 0.0037 and 0.0035. Changes in perfusion induced by adrenergic drugs produced changes in hemoglobin oxygen saturation and [Hb] that correspond with measured alterations of systemic arterial pressure and aortic blood flow. We conclude that diffuse reflectance spectroscopy reliably provides data on intrapancreatic hemoglobin oxygen saturation and [Hb] that can be a valuable tool for minimally invasive on-line evaluation of these aspects of pancreatic perfusion in the rat. This newly designed device is superior to previously used ones in that it analyzes the entire spectrum and therefore can account for changes in scattering that are very likely to occur with pathophysiological alterations such as edema formation.