Temporal heterogeneities in tumor blood supply were studied by using a recently developed first-pass imaging technique. First-pass imaging movies of A-07-GFP human tumor xenografts growing in window chambers were recorded at a frame rate of approximately 9 fps and a spatial resolution of 10.8 x 10.8 microm(2) after a bolus of 155-kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. Each tumor was subjected to imaging thrice, with 20 minutes between each repetition. Highly specific maps of the vascular network and blood supply time (BST) images (i.e., images of the time from when arterial blood enters a tumor through the main supplying artery until it reaches a vessel segment within the tumor) were produced from the movies. The tumors had one to three supplying arterioles and showed substantial temporal heterogeneity in BST. Homogeneous changes in BST in the entire vascular network were seen in tumors supplied by one arteriole. Blood supply time fluctuations in tumor subregions were observed in tumors having two or three supplying arterioles. In addition, individual vessel segments frequently showed significant changes in BST with time. High-magnification transmission microscopy imaging substantiated that BST changes could be a consequence of arterial/arteriolar vasomotor activity, vessel wall compression, varying flow rate, and vascular stasis.