BACKGROUND In the absence of a US Food and Drug Administration (FDA)-cleared latex skin testing reagent, in vitro tests remain important for the diagnosis of latex allergy. OBJECTIVE To evaluate the performance characteristics of IMMULITE 2000 3gAllergy (Immulite), a third-generation, FDA-cleared, continuous random-access immunoanalyzer, for the quantification of latex specific IgE. METHODS Stored serum samples (N = 201) from patients classified as having positive or negative latex puncture skin test results were measured for latex specific IgE levels using Immulite, and these data were compared with historical results from 3 second-generation, FDA-cleared IgE antilatex assays (AlaSTAT [Ala], AutoCAP [CAP], and HY*TEC enzyme immunoassay [HT]). RESULTS The diagnostic performances of the CAP, Ala, and Immulite assays (> or = 0.35 kU/L cutoff value) were equivalent in sensitivity and specificity (P > .05). The HT assay (> or = 0.05 kU/L cutoff value) was more sensitive and less specific (P < .05). Immulite (> or = 0.10 kU/L cutoff value) had greater sensitivity than Ala and CAP and greater specificity than HT (P < .05 for both). Diagnostic efficiency was greater for Immulite than for CAP, Ala, and HT (P < .05). CONCLUSIONS The Immulite system is superior in diagnostic performance, especially at the 0.10 kU/L or greater cutoff level, for the diagnosis of latex allergy compared with older, second-generation assays. Immulite still misclassifies 15.5% of puncture skin test-positive individuals as negative for latex specific IgE. Compared with second-generation assays, Immulite represents a technological advance, with enhanced speed and less operator intervention.