Conventional epifluorescence microscopy coupled with chronic animal window models has provided stunning insight into tumor pathophysiology, including gene expression, angiogenesis, interstitial transport, and drug delivery. However, the findings to date have been limited to the tumor surface (<150 microns). This is an important drawback because the internal architecture of tumors is known to be heterogeneous, with a collagenous tumor/host interface, highly vascularized outer regions, and poorly vascularized inner necrotic regions. Here we present the first application of the multiphoton laser-scanning microscope (MPLSM) to monitoring drug delivery in tumors, phenotypic tumor cell behavior, and tumor-induced gene promoter activity in vivo. Furthermore, we show that the MPLSM can be used in living tumors to quantify physiological parameters such as vascular density, blood flow velocity, leukocyte/endothelial interactions, and single vessel permeability. These measurements are performed with high three-dimensional resolution up to depths of several hundred microns, thus providing novel insights into the internal milieu of tumors. These findings will allow the development of drug therapeutic strategies that not only affect the tumor surface, but also internal regions.