Administration of dexamethasone (DEX) to cattle is commonly used in models of stress-induced effects on host defense, including models investigating interactions of microorganisms with their host. Much less is known about the effects of DEX on the adaptive immune response in cattle compared with other species. The objective of the present study was to characterize subsets of circulating lymphocytes in calves before and 48h after the onset of parenteral DEX treatment. Treatment significantly reduced the overall percentage of circulating lymphocytes and disproportionately depleted the population of γδTCR+/CD8α− cells. Analysis within the CD8α+ population of T cells revealed that DEX treatment also reduced the CD8αlow subset of γδT cells coexpressing the activation marker ACT-2+. By contrast, DEX treatment did not affect the percentage of CD8αlow/CD25+ cells, indicating that cells with a special activation state were affected. Dexamethasone treatment reduced the number of γδT cells but increased the percentages of CD14+ monocytes and activated CD25+ cells (both CD4− and CD4+) in peripheral blood mononuclear cell (PBMC) preparations. Although DEX treatment reduced the overall proliferative capacity of PBMC, it enhanced the relative number of proliferating CD4+ lymphocytes. Lower levels of mRNA for several Th-prototype cytokines (IL-2, IFN-β, IL-4, transforming growth factor-β) were detected in short-term PBMC cultures established from DEX-treated calves compared with PBMC cultures from control calves; the amount of il-10 transcripts, however, was unaffected. Results of the study reported here clearly show that DEX treatment does not uniformly suppress the bovine immune system but has differential effects on lymphocyte sub-populations and functions. This information must be considered when using DEX treatment as a bovine stress model.