CD5⁺ is a cell surface molecule involved in antigen recognition and is present on all T cells and a subset of B cells. Recent work in our laboratory on cattle infected with Mycobacterium avium subsp. paratuberculosis (Johne's disease) has shown the differential expression of the CD5 marker on peripheral blood B cells, with a shift in B cell expression from CD5^dim to CD5^bright as disease progresses from a subclinical to a clinical state. Previous research has also shown an increase in the number of peripheral blood B cells and a decrease in peripheral blood CD4⁺ T cells as disease progression occurs. The purpose of this study was to examine lymphocyte subsets and CD5⁺ expression on peripheral blood B and T cells from healthy cattle and cattle in the subclinical and clinical stages of paratuberculosis. Peripheral blood mononuclear cells (PBMC) were isolated, cultured in the presence or absence of live M. paratuberculosis, and then analyzed by flow cytometry within the three treatment groups. Three CD5⁺ populations were identified: CD5^dim, CD5^bright, and CD5^{extra bright}. Analysis showed an increase in B cells in clinical animals compared to subclinically infected cows. In addition, a decrease in the CD5^dim B cell population along with a concomitant increase in the CD5^bright B cell population was observed in subclinically and clinically infected cows. No significant trends were observed in T cell populations or CD5⁺ expression within T cell populations, and in vitro infection with live M. avium subsp. paratuberculosis did not result in significant changes in B cell or T cell populations. These results suggest that changes in CD5⁺ expression on B cells in animals with paratuberculosis may play a role in progression of the disease.