Brown spider (Genus Loxosceles) bites are normally associated with necrotic skin degeneration, gravitational spreading, massive inflammatory response at injured region, platelet aggregation causing thrombocytopenia and renal disturbances. Brown spider venom has a complex composition containing many different toxins, of which a well-studied component is the dermonecrotic toxin. This toxin alone is able to reproduce mostly toxic effects triggered by the crude venom. Biochemically, dermonecrotic toxin belongs to a family of toxins with 30-35 kDa characterized as sphingomyelinase-D. From cDNA library of L. intermedia venom gland, we cloned and expressed two recombinant isoforms of the dermonecrotic toxin LiRecDT2 (1062pb cDNA) and LiRecDT3 (1007pb cDNA) that encode for signal peptides and complete mature proteins. Phylogenetic tree analysis revealed a structural relationship for these toxins compared to other members of family. Recombinant molecules were expressed as N-terminal His-tag fusion proteins in Escherichia coli and were purified to homogeneity from cell lysates by Ni²⁺ chelating chromatography, resulting in proteins of 33,8kDa for LiRecDT2 and 34,0kDa for LiRecDT3. Additional evidence for related toxins containing sequence/epitopes identity comes from antigenic cross-reactivity using antibodies against crude venom toxins and antibodies raised with a purified dermonecrotic toxin. Recombinant toxins showed differential functionality in intradermal injection rabbits: LiRecDT2 caused a macroscopic lesion with gravitational spreading, while LiRecDT3 evoked transient swelling and erythema. Light microscopic analysis of skin biopsies revealed edema, a collection of inflammatory cells in and around blood vessels and a proteinaceous network at the dermis. Moreover, differential functionality was also demonstrated by a high sphingomyelinase activity for LiRecDT2 and low activity for LiRecDT3 as well as greater in vitro platelet aggregation and blood vessel permeability induced by LiRecDT2 and residual activity for LiRecDT3. Cloning and expression of two recombinant dermonecrotic toxins demonstrate an intraspecific family of homologous toxins that act in synergism for deleterious activities of the venom and open possibilities for biotechnological applications for recombinant toxins as research tools for understanding the inflammatory response, vascular integrity and platelet aggregation modulators.