In breast cancer patients, radiation therapy planning must account for individual anatomy to ensure optimal coverage of tumor and internal mammary nodes. To achieve this, three-dimensional radionuclide lymphoscintigraphy (RNLS) was performed in 167 patients by obtaining two images of the nodes using a 30-degree slant hole collimator rotated 180 degrees between images. Analysis of 768 nodes (mean 4.6/patient) visualized from the level of rib 1 through interspace 5 was performed. The number of nodes seen was not a function of patient age. Cross-communication to the contralateral node chain occurred in 13.8% of cases. Eighty-two percent of nodes were located near the first three ribs and interspaces; 23% were located beyond 3.0 cm from the mid-sternal line. At the level of the radiation beam match line (second rib or interspace), 4.5% of nodes were deeper than 3.0 cm. From rib 3 through interspace 5, 3.9% were deeper than 3.0 cm. Using an idealized tangential field, at least one node would have been missed in 16.2% of patients. Three-dimensional RNLS allows definition and localization of normal sized nodes and ensures that radiation therapy portals can be tailored for each individual under treatment.