The MLL gene, located at chromosome 11q23, is fused to a variety of partner genes through chromosomal translocations in 5–10% of acute leukemias. Partial tandem duplication (PTD) of MLL gene (MLL-PTD) has been described in 10% of AML with normal karyotype. Recently, 2-hit model of leukemogenesis has been proposed for AML. However, the cooperating mutations with MLL translocations (MLL-T) or MLL-PTD have not been systematically analyzed. In the present study, we aimed to identify the fusion partners of MLL and to analyze the cooperating mutations, including FLT3 activation mutations, N-ras and CEBPα mutations in de novo AML with MLL rearrangements. The correlation between MLL fusion transcripts and clinicohematological features was also analyzed. Southern blot analysis identified 92 patients with MLL rearrangements. Their ages ranged from one day to 84 years; 44 were male. The distribution of FAB subtypes was 4 M0, 19 M1, 19 M2, 22 M4, 25 M5, 1 M6, and 2 M7. Standard RT-PCR or multiplex RT-PCR followed by Genescan analysis and/or direct sequencing, was used to detect the common MLL fusion transcripts. MLL-PTD was detected in 46 (50.0%), MLL-AF9 in 13, MLL-AF10 in 9, MLL-AF6 in 8, MLL-ELL in 7, MLL-ENL in 2, and MLL-AF1 and MLL-AF4 in one patient each. In addition, 5 rare MLL fusion transcripts, including MLL-LCX, MLL-SEPT6, MLL-CBL, MLL-MSF and MLL-LARG in one patient each, were characterized by cDNA panhandle PCR and/or long distance inverse PCR. Cytogenetic findings were available in 76 patients with MLL rearrangements, 11q23 abnormalities were detected in 27 patients. By PCR-Genescan analysis and direct sequencing, FLT3-ITD mutations were detected in 21 patients with MLL rearrangements. By PCR-RFLP and sequencing, FLT3-TKD mutations were detected in 12 patients. By DNA PCR and direct sequencing, CEBPα and N-ras mutations were found in 1 and 9 patients, respectively. Coexistence of FLT3-ITD and FLT3-TKD mutations was observed in 2 patients, FLT3-ITD and CEBPα mutations in one patient, and FLT3-TKD and N-ras mutations in another one patient. Taken together, cooperating mutations of FLT3 and/or N-ras mutations occurred in 42% (39/92) of AML with MLL rearrangements. The frequency of FLT3-ITD was significantly higher in patients with MLL-PTD than those with MLL-T (P<0.001). There was no difference in the mutation status of FLT3-TKD or N-ras between MLL-PTD and MLL-T groups. Sixty patients received standard induction chemotherapy, 42 achieved a complete remission. The 5-year overall survival and relapse-free survival rates were 14.9% and 27.5%, respectively. The complete remission rate in MLL-PTD group was 56.5% (13/23) compared with 78.4% (29/37) in MLL-T group (P=0.089). Patients with MLL-PTD had a poorer 5-year survival rate than MLL-T group (0% vs. 21.9%, P=0.0623). There was no difference in relapse-free survival between the two groups (P=0.3774). In summary, the fusion partners of MLL were characterized in de novo AML. We have identified 5 rare MLL partner genes, MLL-PTD was the most common genetic subtype. MLL-PTD was highly associated with FLT3-ITD mutations. The finding of high incidence of coexistence of FLT3 or N-ras mutations in AML with MLL rearrangements supports the two-hit hypothesis for the pathogenesis of AML.