The aim of this study was to compare the ability of laying hen abdominal macrophages during the second production cycle by using two different methods of induced molting. Two groups of Single Comb White Leghorn hens were induced to molt at the end of their first production cycle using feed restriction and ZnO supplementation. Macrophages were isolated from the abdomen and in vitro cytotoxic ability, at which point macrophage bactericidal moiety nitric oxide (NO) was recorded. Serum IgM and IgG titers against sheep red blood cells (SRBC) were determined at various stages: before molting (BM), 5% production level (5P), peak production stage (PP) and at the end of production (EP) level after fast and Zn-induced molt. Macrophages adherence percentage remained unaffected (p< or =0.05) during all production cycles, whereas the macrophage engulfment percentage and engulfment/cell was significantly higher (p< or =0.05) at PP in both fast and Zn-induced molted groups, as compared to all other studied stages. Macrophage NO production was increased (p< or =0.05) at PP and after SRBC and lipopolysaccrides (LPS) stimulus, when molted with ZnO supplementation. Serum total antibody titer against SRBC increased serum IgG and IgM titers during the second production cycle by Zn-induced molt. However, molting stress greatly reduced IgG and IgM production at the 5P stage. Serum Zn concentration increased with the onset of production but decreased at the EP stage irrespective of their molting regimes. Our results validate the strengthened innate and acquired immune response during the second production cycle after Zn-induced molting instead of fasting.