Contaminants can cause detrimental effects in wild birds. However, these effects are difficult to measure in all but the most severe cases. Immune function is a sensitive and meaningful biological marker of contaminant-induced effects in captive birds but has more limitations in wild birds due in part to the lack of a proven blood preservation method. We developed methods to assess ex vivo immune function in wild birds using cryopreserved peripheral white blood cells (WBCs). We assessed the effects of cryopreservation on WBC viability and functionality in two immunoassays (concavalin A-induced T lymphocyte proliferation and macrophage phagocytosis) in domestic chickens (Gallus spp.: white Wyandottes and Dominiques) and validated this approach on cryopreserved WBC samples from wild American coots (Fulicia americana). Cryopreservation of chicken WBCs caused a slight but significant decrease in cell viability (99% +/- 0.2 SE for fresh cells versus 84% +/- 2 SE for cryopreserved cells, p = 0.001, Mann-Whitney U, n = 8). No difference was detected in viability between cells that were cryopreserved for less than 10 days (88% +/- 3.7 SE) and more than 50 days (89% +/- 1.3 SE) (n = 6). Overall, there was no statistical difference in the performance of cryopreserved cells compared to fresh cells. Across multiple experiments, cryopreserved T lymphocytes exhibited 200-900% stimulated proliferation above nonstimulated cells, and 40-80% of cryopreserved macrophages ingested yeast. 9,10,Dimethyl-1,2-benz-anthracene (DMBA) reduced proliferation and phagocytosis in cryopreserved cells over an ex vivo exposure range of 0-170 microM DMBA. Tests of immune function on American coot WBCs cryopreserved for up to 10 months (viability of 72% +/- 2.5 SE, n = 24) were similar to the cryopreserved chicken WBCs. This study will facilitate greater use of ex vivo immune function assays as tools to study effects of contaminant exposure in wildlife by demonstrating the viability and functionality of cryopreserved avian cells.