Despite the weak immunogenicity of gangliosides, a limited number of highly specific murine monoclonal antibodies (MAbs) were elicited. This study investigated the reactivity and the structure of the VH and V kappa genes of nine hybridomas obtained from independent fusions producing antibodies against disialogangliosides GD2 and GD3 and their O-acetylated derivatives. These antibodies depended on four types of V kappa genes. They were also encoded by VH genes of the J558 family (5 out of 9) and occasionally by VH genes of the S107, 7183, and 3609 families, rearranged with a variety of DH and JH genes. The 8B6 and 7H2 MAbs specific for GD2-O-acetylated, respectively, used the VH gene of the S107 and 7183 families. The length of H chain CDR3 ranged from 8 to 11 amino acids. A set of S107 and 3609 germline genes closed from A/J murine fetal liver and matched with the VH segment of hybridomas 8B6 and 10B8 revealed somatic mutations. Although the relative number of sequences does not preclude any formal conclusions regarding the preferential use of V genes in the immune recognition of carbohydrate structures, our results clearly indicate that MAbs directed to very similar structures as GD2 and GD3 were encoded by different VH and V kappa genes.