Johne's disease (JD) is characterized by a protracted period of subclinical infection. Infected cows may remain in the subclinical state until stressors such as parturition and lactation invoke more clinical signs of disease. The objective of this study was to evaluate changes in the percentages of CD4(+), CD8(+), and gammadelta T-cells, B-cells, monocytes, as well as the expression of the activation marker, CD5, on these cell subpopulations in the peripheral blood of dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis (MAP) during the periparturient period. Peripheral blood mononuclear cells (PBMCs) were collected from 3 wk pre- to 4 wk post-calving and freshly isolated or cultured for 7d. Day 7 cultures were infected with live MAP at a 10:1 MOI (bacteria to adherent PBMC), and cultures were incubated for an additional 24h. Fluorescent antibody labeling of lymphocyte subsets and monocytes was conducted and analyzed with flow cytometry. Freshly isolated PBMCs from subclinical cows expressed a greater (P<0.05) percentage of CD8(+) and gammadelta T-cells compared with clinical cows. The percentage of CD4(+) T-cells increased (P<0.08) in clinical cows as parturition approached. During the postpartum period, clinical cows had greater (P<0.05) CD4:CD8 ratios compared with subclinical and control cows. After 8d, uninfected PBMCs from clinical cows had greater (P<0.05) percentages of CD14(+) cells compared with subclinical cows. When infected with live MAP, there was no effect of infection group or parturition on cell subpopulations. In fresh PBMCs, clinical cows expressed lower percentages of CD4(+)CD5(bright) and CD8(+)CD5(bright) compared with control cows, but greater percentages of CD5(dim) cells for all lymphocyte subsets. These results suggest changes in the percentages of lymphocyte subsets, monocytes, and CD5 markers are modulated by both infection status and the periparturient period.