High-dose methotrexate (HDMTX) chemotherapy is generally accepted as an effective method for the treatment and prevention of extramedullary leukemia in children. However, it is unknown whether HDMTX chemotherapy kills intestinal bacteria on a large scale, thus causing dysbacteriosis, which may in turn influence the progress or prognosis of leukemia. The aim of this study was to examine changes in intestinal flora in children with acute lymphoblastic leukemia (ALL) treated with HDMTX chemotherapy. Bacterial DNA in stool from 36 healthy children and 36 ALL children were tested at A(260) with a spectrophotometer before and after HDMTX chemotherapy. The primers of Bifidobacteria, Lactobacillus and Escherichia coli were designed according to the 16SrRNA/DNA bacterial sequences. Bacteria were qualitatively and quantitatively confirmed by routine polymerase chain reaction (PCR) and fluorescent quantitative PCR, respectively. Our data showed that the total amount of flora in the stools of children with ALL was decreased by 29.6% compared with healthy children (P<0.01). The total amount of flora in the stools of children with ALL on the third and seventh days after chemotherapy were 1496.5 +/- 577.1 and 1966.6 +/- 598.3 ng/mu L, respectively, which was notably less than before chemotherapy (2436.3 +/- 768.6 ng/mu L). The amount of Bifidobacteria, Lactobacillus and E. coli in the intestinal tract in the ALL group after chemotherapy had an apparent change, which decreased most clearly on the third day, and partially recovered on the seventh day after chemotherapy. HDMTX chemotherapy can cause intestinal dysbacteriosis in children with ALL. The amount of Bifidobacteria, Lactobacillus and E. coli decreased significantly compared with the control group.