The study was aimed to investigate the association of FOXP3 gene expression in donor grafts with acute graft-versus-host disease after HLA-identical sibling allogeneic hematopoietic stem cell transplantation. Twenty-six donor grafts (peripheral blood or bone marrow) and their respective clinical characteristics were evaluated. Flow cytometry analysis was performed to assess the percentage of CD4+CD25+ and CD4+CD25(high) T cells in cord blood, healthy controls' peripheral blood and donor grafts. Relative transcripts of FOXP3 mRNA were determined by real-time quantitative reverse transcription -polymerase chain reaction with beta2-MG as the internal control gene. The specificity of FOXP3 and beta2-MG amplifications was confirmed by analyzing the dissociation curves and electrophoresis of the target amplicon. The results showed that the CD4+CD25+ T cells in peripheral blood, peripheral blood stem cell (PBSC) or BM grafts exhibited a continuous and primarily low expression of CD25 and the frequencies of CD4+CD25+ T and CD4+CD25(high) T in CD4+ T cells were (48.5 +/- 16.3)% and (9.6 +/- 2.5)%, (42.1 +/- 14.7)% and (13.1 +/- 4.2)%, (43.4 +/- 9.6)% and (14.6 +/- 4.5)%, respectively. There was no significant difference in the frequencies and absolute numbers of CD4+CD25(high) T cells between patients with aGVHD and patients without aGVHD (P > 0.05). The plot of log transfused cDNA amount versus DeltaCt had a slope of 0.0826 which indicated approximately equal efficiency of FOXP3 and beta2-MG amplifications in real-time PCR. The specificities of amplification were confirmed by analyzing the dissociation curves and electrophoresis of PCR products with the values of Tm 86.5 degrees C and 82.3 degrees C, respectively. The relative transcripts of FOXP3 in PBSC grafts of recipients without aGVHD were 318%high as those with aGVHD (median of 41.0 x 10(-5) and 12.9 x 10(-5), respectively) (P = 0.03). No significant difference was found in other related variables for GVHD. It is concluded that coexpression of CD4 and CD25 may be insufficient to identify regulatory T cells; FOXP3 mRNA expression may be specifically quantified with real-time quantitative RT-PCR using SYBR Green I chemistry. FOXP3 mRNA expression in donor grafts is significantly low in patients with aGVHD compared with patients without aGVHD. It indicated that the expression level of FOXP3 mRNA may be one of the useful indicators for in predicting aGVHD.