During the effector phase of graft-versus-host disease (GvHD) response, donor T cells play an essential role and they are believed to change the expression of activation and co-stimulatory markers associated with functional alloreactivity. We analysed the expression of CD25, CD69, HLA-DR, CD154 and CD134 on CD4⁺ and CD8⁺ T cells by flow cytometry during acute GvHD (aGvHD) in 24 patients receiving human leucocyte antigen (HLA)-identical stem cell transplants. Expression of these molecules in nine patients with stages I–IV aGvHD was compared with 15 patients without aGvHD (n = 15). Serial analysis showed that peripheral blood of aGvHD patients presented a significant increase of CD4⁺ CD25⁺ cells (P < 0.03), CD4+ CD69⁺ (P < 0.04) and CD4⁺ CD134⁺ cells (P < 0.01). Additionally, there was a significant increase in CD8⁺ cells expressing CD134 (P = 0.007) and CD154 (P = 0.02). After resolution of aGvHD, the increased expression of these molecules returned to values comparable to patients without aGvHD. Only two of the 15 patients without clinical signs of aGvHD presented activated T cells that could not be attributed to development of aGvHD. In summary, our data show that multiple activation molecules are preferentially up-regulated on CD4⁺ and CD8⁺ T cells from patients with aGvHD. These patients had a significant increase in the expression of the co-stimulatory molecules CD134 and CD154.