Mitochondrial transfer RNA (mt-tRNA) mutations have been reported to be associated with a variety of diseases. In a previous paper that studied the mtDNA background effect on clinical expression of Leber's hereditary optic neuropathy (LHON) in 182 Chinese families with m.11778G>A, we found a strikingly high frequency (7/182) of m.593T>C in the mitochondrially encoded tRNA phenylalanine (MT-TF) gene in unrelated LHON patients. To determine the potential role of m.593T>C in LHON, we compared the frequency of this variant in 479 LHON patients with m.11778G>A, 843 patients with clinical features of LHON but without the three known primary mutations, and 2374 Han Chinese from the general populations. The frequency of m.593T>C was higher in LHON patients (14/479) than in suspected LHON subjects (12/843) or in general controls (49/2374), but the difference was not statistically significant. The overall penetrance of LHON in families with both m.11778G>A and m.593T>C (44.6%) was also substantially higher than that of families with only m.11778G>A (32.9%) (P = 0.083). Secondary structure prediction of the MT-TF gene with the wild type or m.593T>C showed that this nucleotide change decreases the free energy. Electrophoretic mobility of the MT-TF genes with the wild type or m.593T>C transcribed in vitro further confirmed the change of secondary structure in the presence of this variant. Although our results could suggest a modest synergistic effect of variant m.593T>C on the LHON causing mutation m.11778G>A, the lack of statistical significance probably due to the relatively small sample size analyzed, makes necessary more studies to confirm this effect.