A transforming N-ras allele was molecularly cloned from the RD human rhabdomyosarcoma cell line, and the nature of its activation studied. Construction of chimeric recombinants between the RD-transforming allele and a normal human allele enabled us to localize the alteration responsible for the activation to the second exon of the N-ras gene. The nucleotide sequence of this exon, when compared to that of the normal allele, revealed a single difference at the 61st amino acid position of the encoded protein; the CAA codon for glutamine in the normal allele was mutated to a CAT codon for histidine in the RD-transforming allele. This result is the first description of a histidine replacing glutamine in the 61st position and provides further evidence that the 61st amino acid is one of the preferential sites for N-ras activation.