The aim of this study was to analyze the influence of aerobic fitness (AF) on age-related lymphocyte DNA damage in humans, giving special attention to the role of the mitochondrial respiratory chain and hydrogen peroxide production. Considering age and AF (as assessed by VO(2)max), 66 males (19-59 years old) were classified as high fitness (HF) or low fitness (LF) and distributed into one of the following groups: young adults (19-29 years old), adults (30-39 years old), and middle-aged adults (over 40 years old). Peripheral lymphocytes obtained at rest were used to assess DNA damage (strand breaks and formamidopyrimidine DNA glycosylase (FPG) sites through the comet assay), activity of mitochondrial complexes I and II (polarographically measured), and the hydrogen peroxide production rate (assayed by fluorescence). Results revealed a significant interaction between age groups and AF for DNA strand breaks (F = 8.415, p = .000), FPG sites (F = 11.766, p = .000), mitochondrial complex I activity (F = 7.555, p = .000), and H(2)O(2) production (F = 7.500, p = .000). Except for mitochondrial complex II activity, the age variation of the remaining parameters was significantly attenuated by HF. Considering each AF level, an increase in DNA strand breaks and FPG sites with age (r = 0.655, p = 0.000, and r = 0.738, p = 0.000, respectively) was only observed in LF. Moreover, decreased mitochondrial complex I activity with age (r = -.470, p = .009) was reported in LF. These results allow the conclusion that high AF seems to play a key role in attenuating the biological aging process.