In order to study the possibility of using lymphocyte subsets as a stress indicator, the changes of the subset proportion in peripheral blood were examined after transportation. Six 3-month-old male Holstein calves were transported for about 3 h in a lorry via an ordinary road. Blood samples were collected at the expected unloading time on the day before transport (T0), immediately after unloading (T1), 24 h after unloading (T2), and 1 week after unloading (T3). CD2, CD4, CD8, CD21, CD25, WC1, and NKp46 antigens on peripheral blood mononuclear cells were measured by flow cytometry. There was an increase in the proportions of CD25+ cells and WC1+ γδT cells at T1 and T2. The proportions of NK cells increased at T1, including an increase in the population of CD2- NK cells. CD2+ NK cells did not show any change. The proportions at T1 were lower than at T3 in CD21+ B cells. CD4+ and CD8+ αβT cells did not change significantly, although CD2+ T cells showed a trend toward reduction at T1. Consequently, CD2- NK cells and WC1+ γδT cells might offer potential as stress index for calf transport.