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Honey bee, Apis mellifera L., hygienic behavior is a mechanism of disease resistance and a mode of defense against the parasitic mite Varroa jacobsoni Oudemans. Hygienic bees uncap and remove diseased and parasitized brood from the nest. The propagation of colonies that demonstrate resistance to chalkbrood and American foulbrood and that remove pupae infested by Varroa mites is becoming increasingly important in apiculture. This study evaluates 2 commonly used field assays used to screen colonies for hygienic behavior: the freeze-killed brood and the pierced brood assays. Both involve determining the time required for worker bees to remove dead capped brood from a section of comb. Colonies in the experiment displayed a wide range of removal rates and were grouped as hygienic, nonhygienic, or intermediate. The results of experiments 1 and 2 indicated that neither the age nor the source of the frozen brood had a significant effect on the removal rate by hygienic colonies (i.e., those colonies that consistently uncapped and removed freeze-killed brood within 48 h). In experiment 3, only a weak correlation was found between the removal of young freeze-killed and pierced pupae, but a significant correlation existed between the removal of pre-eclosion freeze-killed and pierced pupae. Experiment 4 examined cues that elicit removal behavior by hygienic and non hygienic colonies. When pupae were pierced with an insect pin through the base of the cell (without piercing the wax cell capping), there was no difference in the number of pupae removed by the hygienic and nonhygienic colonies. On average, 30% of all pierced pupae survived the treatment, which considerably diminished the accuracy and reproducibility of the test. When pupae were treated with hemolymph extracted from either a live or freeze-killed pupa, there was also no difference in the rate of removal by hygienic and nonhygienic colonies. These results indicate that bees from nonhygienic lines can be induced to express hygienic behavior only if a sufficiently strong stimulus is present. Both hygienic and nonhygienic colonies removed significantly more pupae treated with hemolymph from a dead pupa than hemolymph from a live pupa, indicating that the cue that stimulates removal behavior is stronger in dead pupae. It is concluded that the freeze-killed brood assay is the most conservative and reliable screening procedure for hygienic behavior. The following procedures are recommended: Randomly selected comb sections (5 by 6 cm each) of capped brood should be cut from 1 healthy colony, frozen, and introduced into the test colonies. The assay should be repeated at least twice. Only colonies that remove >95% of freeze-killed brood within 48 h in both tests should be considered hygienic. When developing hygienic breeder stock, the hygienic colonies should be challenged with the American foulbrood or chalkbrood pathogen to ensure resistance.