Pure venoms from yellow jackets and bees were used to evaluate denaturing and nondenaturing immunoblot techniques and to compare results of IgE antibody-stained immunoblots to RAST with highly purified allergens. Significant differences in IgE antibody binding were observed among all three techniques, suggesting that tertiary conformation is a major factor in the allergenic determinants of venom allergens. IgG antibodies from hyperimmunized animals and from specimens obtained from allergic patients before and after venom immunotherapy appeared to exhibit less conformational dependence for binding. The conformational dependence of IgE antibody binding to bee and yellow jacket phospholipases was confirmed by direct and inhibition RAST studies. The allergens studied were all single polypeptide chains.