Impairment of cochlear blood flow has been discussed as factor in the pathophysiology of various inner ear disorders. However, the microscopic study of cochlear microcirculation is limited due to small scale and anatomical constraints. Here, two-photon fluorescence microscopy is applied to visualize cochlear microvessels. Guinea pigs were injected with Fluorescein isothiocyanateor Texas red-dextrane as plasma marker. Intravital microscopy was performed in four animals and explanted cochleae from four animals were studied. The vascular architecture of the cochlea was visualized up to a depth of 90.0 +/- 22.7 mu m. Imaging yielded a mean contrast-to-noise ratio (CNR) of 3.3 +/- 1.7. Mean diameter in vivo was 16.5 +/- 6.0 mu m for arterioles and 8.0 +/- 2.4 mu m for capillaries. In explanted cochleae, the diameter of radiating arterioles and capillaries was measured with 12.2 +/- 1.6 mu m and 6.6 +/- 1.0 mu m, respectively. The difference between capillaries and arterioles was statistically significant in both experimental setups (P < 0.001 and P = 0.022, two-way ANOVA). Measured vessel diameters in vivo and ex vivo were in agreement with published data. We conclude that two-photon fluorescence microscopy allows the investigation of cochlear microvessels and is potentially a valuable tool for inner ear research.