Background Allergen-specific IgE (sIgE) measurements are used to help identify causative allergenic agents and to determine the degree of sensitization to facilitate treatment decisions. We examined the performance of a new third-generation chemiluminescent enzyme immunoassay for allergen-specific IgE (sIgE) on the continuous random access Immulite® 2000 system. Methods Detection limit and dilutional linearity were determined. Within-run and total precision were assessed according to the NCCLS EP5 guideline. Interlaboratory comparison of the new Immulite 2000 third-generation allergen-specific IgE assay was performed, as well as intermethod comparison against the Pharmacia FEIA, a second-generation assay. Results The detection limit was < 0.1 kU/l. Dilutional linearity held from 100 down to 0.2 kU/l. Regression analysis of the interlaboratory comparison results yielded: Immulite 2000(Laboratory 1) = 1.07 Immulite 2000(Laboratory 2) + 0.18 kU/l; r = 0.98, n = 3588 results. Intermethod comparison showed the following: Immulite 2000 = 0.83 (Pharmacia FEIA) + 0.42 kU/l; r = 0.79, n = 512 results. Bland–Altman analysis of the interlaboratory and intermethod comparisons indicated no systematic bias. Conclusions We confirmed the reported performance characteristics of the third-generation sIgE assay and found reasonably good interlaboratory and intermethod agreement. The extended range capability of the third-generation assay provides a new tool for investigating cutoffs and trends in childhood allergy disease progression at concentrations < 0.35 kU/l.