We tested the hypothesis that exercise could stimulate CD34+ peripheral blood hematopoietic stem cells (PBSC) in children. Fourteen early pubertal boys (EP, age 10.3 +/- 0.3 y) and 13 late pubertal boys (LP, age 16.5 +/- 0.4 y) performed 20 min of moderate-to-vigorous cycle ergometer exercise. Blood was drawn before and after exercise. Cells were stained for surface CD34+. Plasma granulocyte colony stimulating factor (G-CSF), Fms-like tyrosine kinase-3 (FLT-3), and stromal cell-derived factor-1 (SDF-1) were measured using ELISA. Exercise substantially increased PBSC (in EP from 112 +/- 21 to 182 +/- 30 cells/microL, p=0.0007; in LP from 63 +/- 8 to 152 +/- 21, p=0.0008), and to a smaller extent FLT-3 (in EP from 98 +/- 5 to 110 +/- 6 pg/mL, p<0.0001; in LP from 73 +/- 6 to 92 +/- 6, p<0.0001) and G-CSF (in EP from 26 +/- 4 to 29 +/- 4 pg/mL, p<0.0001; in LP from 14 +/- 1 to 18 +/- 1, p<0.0001). Baseline levels of PBSC, FLT-3, and G-CSF were significantly higher in EP. Exercise increased SDF-1 alpha only in LP, and the FLT-3 increase was greater in LP than EP. Brief exercise affects PBSC and PBSC mediators in children.