To provide investigative tools for the study of neuroblastoma (NB) biology and therapy, we have characterized five orthotopic (adrenal) human xenograft models of NB. Initial experiments compared subcutaneous (heterotopic) with adrenal (orthotopic) injections of two NB cell lines (SK-N-AS and SMS-KCNR) in Beige-SCID mice. These studies demonstrated more relevant tumor biology, including angiogenic phenotype, and enhanced spontaneous distant metastasis for orthotopic versus heterotopic tumors. RNase protection assay demonstrated differences in the expression of angiogenesis-associated genes (flt1, TIE1, angiopoietin, and endoglin) between adrenal and subcutaneous xenografts. Orthotopic models were used to define and characterize the three remaining NB cell lines (SH-SY5Y, LA-1-15N, and IMR32). The pattern of angiogenesis was distinctive for each xenograft model and included a variety of vascular structures. The sites for metastases were distinct for each cell line and included lymph nodes, liver, ovaries, lungs, bone marrow and local bone extension. These well characterized, relevant, highly angiogenic, and metastatic orthotopic models of NB will be a valuable resource to improve our understanding of the biology and treatment of NB.