We characterized the antigenic cross-reactivity of two medically important North American Loxoxceles species: L. reclusa (native to southeastern US) and L. deserta (native to southwestern US). Dermonecrosis resulting from bites from these two North American spider species are indistinguishable clinically. Polyclonal IgG antivenins directed against L. reclusa and L. deserta were raised in rabbits and used to develop specific enzyme immunoassays (EIAs). Antigenic differences in the two venoms were evaluated as follows: (1) Comparison of the sensitivities and correlation coefficient (R(2)) of anti-L. reclusa (alpha LoxR) and anti-L. deserta antibodies (alpha LoxD) in the detection of varying concentrations of the two venoms; (2) separation and western blot comparison of venom components; (3) protein sequence analysis of L. desertavenom and comparison to the L. reclusa protein sequence analysis present in a US national database; and (4) in vivo evaluation of alpha LoxR and alpha LoxD antivenins in attenuating dermal lesions (rabbit model). Correlation coefficients for alpha LoxR (R(2)=0.99) and alpha LoxD (R(2)=0.99) polyclonal antibodies in the measurements of standard concentrations of venoms were virtually identical. Western blot analysis revealed multiple common bands between the two venoms. Amino acid data (amino acids 1-35, N-terminal) of the active venom components of the two venoms revealed only three non-identical amino acids. alpha LoxR and alpha LoxD antivenins were similarly effective in blocking the development of rabbit skin lesions (ANOVA p<0.05). In summary, L. reclusa and L deserta spider venoms possess several common protein bands as identified by western blot, greater than 90% amino acid sequence identity, and marked antigenic cross-reactivity.