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Background The molecular mechanism of human tetralogy of Fallot (TOF) is incompletely defined. Animal models have suggested that neurotrophic tyrosine receptor kinase 3 (NTRK3) might be associated with the outflow tract defect, similar to that seen in human TOF, however, the expression patt of NTRK3 in human TOF heart tissues has never been investigated. Methods Quantitative real-time PCR (qRT-PCR) and immunohistochemistry were applied to detect NTRK3 mRNA and protein levels in right ventricular outflow tract tissue samples of TOF patients, ventricular septal defect (VSD) patients and normal control infants (n=10 in each group). Results qRT-PCR analysis indicated that NTRK3 mRNA levels were significantly decreased in the TOF group compared to the VSD group (0.024±0.003 vs 0.085±0.004, P=0.022) and the normal control group (0.024±0.003 vs 0.091±0.002, P=0.006). Quantitative immunohistochemical analysis showed that NTRK3 protein was mainly localized in the myocardium cytoplasm in all 3 groups. The immunoreactivity of NTRK3 protein was again significantly lower in the TOF group compared to the VSD group (1.42±0.62 vs 14.12±1.83, P=0.023) and the control group (1.42±0.62 VS 16.25±2.31, P=0.008). The expression of NTRK3 in the VSD group and in the control group showed no significant differences at both mRNA and protein levels. Conclusions Insufficient expression of NTRK3 is associated with the outflow tract defect of human tetralogy of Fallot and may contribute to the progression of this defect.