Little is known about the prevalence and significance of ras gene activation in neural crest tumors such as neuroblastomas, pheochromocytomas, and medullary thyroid cancers (MTCs). Therefore, we analyzed DNA from 10 human neuroblastoma cell lines and 10 primary human pheochromocytomas for activating mutations in N-ras, H-ras, and K-ras. We also studied DNA from 24 primary neuroblastomas and 10 MTCs for N-ras mutations. ras genes were analyzed by direct sequencing of specific DNA fragments amplified by the polymerase chain reaction. With the exception of the SK-N-SH cell line, the examined ras gene sequences were normal in all the neuroblastomas, pheochromocytomas, and MTCs tested. A single point mutation was identified at codon 59 (GCT(ala)----ACT(thr)) in one N-ras allele in an SK-N-SH subline. Interestingly, this mutation is different from the activating codon 61 mutation which resulted in the initial identification of N-ras from SK-N-SH DNA. Therefore, we analyzed the sequences of earlier passages and sublines of the SK-N-SH cell line, but mutations at codon 59 or 61 were not detected, suggesting that neither mutation was present in the primary tumor. Our results indicate that N-ras mutations may occur spontaneously during in vitro passage of cell lines but rarely, if ever, occur in primary neuroblastomas, pheochromocytomas, and MTCs. In addition, we have not found H-ras or K-ras mutations in any neuroblastoma cell line or primary pheochromocytoma.