Activating mutations in the ALK gene were identified in 8% of primary neuroblastomas, with three somatic and two germline mutations. The most common mutation, F1174L, was found in three neuroblastoma cell lines and was sensitive to the ALK inhibitor TAE684. These mutations conferred sensitivity to ALK inhibition, suggesting a molecular basis for targeted therapy. A genome-wide analysis showed high-level ALK amplification in 15% of neuroblastomas with MYCN amplification. ALK mutations were associated with oncogenicity, and functional studies demonstrated that F1174L and R1275Q mutations activated ALK kinase activity, leading to cytokine-independent growth in Ba/F3 cells. TAE684 inhibited ALK activity in these cells, with F1174L being highly sensitive. Neuroblastoma cell lines with F1174L mutations were sensitive to TAE684, while those with R1275Q were resistant. ALK expression levels were lower in TAE684-sensitive cell lines, and inhibition of ALK kinase activity increased ALK protein levels. Resistance in some cell lines may be due to other molecular aberrations. ALK is a potential therapeutic target in neuroblastoma, and further studies are needed to validate its role in patient tumors. The findings suggest that ALK inhibitors may be effective in treating neuroblastomas with activating mutations.Activating mutations in the ALK gene were identified in 8% of primary neuroblastomas, with three somatic and two germline mutations. The most common mutation, F1174L, was found in three neuroblastoma cell lines and was sensitive to the ALK inhibitor TAE684. These mutations conferred sensitivity to ALK inhibition, suggesting a molecular basis for targeted therapy. A genome-wide analysis showed high-level ALK amplification in 15% of neuroblastomas with MYCN amplification. ALK mutations were associated with oncogenicity, and functional studies demonstrated that F1174L and R1275Q mutations activated ALK kinase activity, leading to cytokine-independent growth in Ba/F3 cells. TAE684 inhibited ALK activity in these cells, with F1174L being highly sensitive. Neuroblastoma cell lines with F1174L mutations were sensitive to TAE684, while those with R1275Q were resistant. ALK expression levels were lower in TAE684-sensitive cell lines, and inhibition of ALK kinase activity increased ALK protein levels. Resistance in some cell lines may be due to other molecular aberrations. ALK is a potential therapeutic target in neuroblastoma, and further studies are needed to validate its role in patient tumors. The findings suggest that ALK inhibitors may be effective in treating neuroblastomas with activating mutations.