We have analyzed N-myc gene amplification and N-myc protein expression in 41 primary neuroblastomas. In this series, 22 patients are currently alive and disease-free, whereas 19 patients have died or are alive with progressive disease. All tumor samples were obtained at operation. N-myc gene amplification was detected by Southern blot analysis, and N-myc protein expression was detected using Bouin-fixed, paraffin-embedded tissue sections and immunohistochemical staining with anti-N-myc gene products serum. N-myc protein expression was detected in all 9 tumors with N-myc gene amplification (greater than or equal to 10 N-myc gene copies). Among 19 patients with poor prognosis, N-myc gene amplification was detected in 8 (42%) and N-myc protein expression in 18 (95%); neither was detected in 20 of the 22 patients who survived free of disease. We conclude that the immunohistochemical detection of N-myc protein expression is one of the most unfavorable prognostic factors in neuroblastoma patients.