Diffuse optical tomography (DOT) methods observe hemodynamics in the brain by measuring light transmission through the scalp, skull, and brain. Thus, separating signals due to heart pulsations, breathing movements, and systemic blood flow fluctuations from the desired brain functional responses is critical to the fidelity of the derived maps. Herein, we applied independent component analysis (ICA) to temporal signals obtained from a high-density DOT system used for functional mapping of the visual cortex. DOT measurements were taken over the occipital cortex of human adult subjects while they viewed stimuli designed to activate two spatially distinct areas of the visual cortex. ICA was able to extract clean functional hemodynamic signals and separate brain activity sources from hemodynamic fluctuations related to heart and breathing without knowledge of the stimulus paradigm. Furthermore, independent components were found defining distinct functional responses to each stimulus type. Images generated from single ICA components were comparable, with regard to spatial extent and resolution, to images from block averaging (with knowledge of the block stimulus paradigm). Both images and estimated time-series signals demonstrated that ICA was superior to principal component analysis in extracting the true event-evoked response signals. Our results suggest that ICA can extract the time courses and the corresponding spatial extent of functional responses in DOT imaging. Hum Brain Mapp, 2009. © 2009 Wiley-Liss, Inc.