The whole-blood chemiluminescence (WBCL) assay is a simple and rapid method of measuring production of reactive oxygen species by circulating leukocytes, particularly heterophils (birds) and neutrophils (other vertebrates). In the interest of substantiating a broadly applicable measure of innate immunity, we investigated the microplate WBCL method for several wildlife species as well as domestic broiler chickens. Lucigenin as a light enhancer was used for all avian blood, wild and domestic, while luminol was used in bear and frog blood. Use of ethylenedinitrilo-tetraacetic acid (EDTA) as the anticoagulant caused hemolysis of frog blood and decreased WBCL responses in all animals tested. Heparin, even in high concentrations, caused modest to no decrease of WBCL responses. The WBCL response correlated highly with heterophil or neutrophil numbers in all species tested. The WBCL response was tested in freshly collected blood as well as in blood one to five days postcollection in order to determine the utility of this assay for field studies when immediate access to laboratory facilities is not possible. One to three days of delay in performing the test after blood collection caused no, or only a slight, decay of the chemiluminescence response in most animals. Using domestic chickens, we tested the sensitivity of the WBCL method to detect differences between treatment groups and looked for loss of chemiluminescence response over several days using the original blood samples. Significant differences in the WBCL response between experimental groups of broiler chicken were detectable in freshly collected blood, as well as one- to four-day-old blood. Our results show that the innate immune response of populations of wild animals may be successfully compared using this assay, even when blood cannot be tested until a few days after collection.