The pathophysiology of microcirculation is intensively investigated to understand disease development at the microscopic level. Orthogonal polarization spectral (OPS) imaging and its successor sidestream dark-field (SDF) imaging are relatively new noninvasive optical techniques allowing direct visualization of microcirculation in both clinical and experimental studies. The goal of this experimental study was to describe basic microcirculatory parameters of skeletal muscle and ileal serous surface microcirculation in the rat using SDF imaging and to standardize the technical aspects of the protocol. Interindividual variability in functional capillary density (FCD) and small vessels (<25 microm in diameter) proportion was determined in anesthetized rats on the surface of quadriceps femoris (m. rectus femoris and m. vastus medialis) and serous surface of ileum. Special custom made flexible arm was used to fix the SDF probe minimizing the pressure movement artifacts. Clear high contrast images were analyzed off-line. The mean FCD obtained from the surface of skeletal muscle and ileal serous surface was 219 (213-225 cm/cm(2)) and 290 (282-298 cm/cm(2)) respectively. There was no statistically significant difference between rats in mean values of FCD obtained from the muscle (P = 0.273) in contrast to ileal serous surface, where such difference was statistically significant (P = 0.036). No statistically significant differences in small vessels percentage was detected on either the muscle surface (P = 0.739) or on ileal serous surface (P = 0.659). Our study has shown that interindividual variability of basic microcirculatory parameters in rat skeletal muscle and ileum is acceptable when using SDF imaging technique according to a highly standardized protocol and with appropriate fixation device. SDF imaging represents promising technology for experimental and clinical studies.