In this study we looked at smokers with and without chronic obstructive pulmonary disease (COPD) patients in order to evaluate the incidence of 4977 base pair (bp) mtDNA (mtDNA4977) deletion and mtDNA copy number in sputum cells and in peripheral blood leukocytes (PBLs) in relation to mitochondrial function and oxidative stress status. Twenty-five COPD patients who were current smokers, 22 smokers and 23 healthy nonsmokers (for only PBLs studies) participated in this study. The 4977-bp deletion was detected in all examined samples within 40 cyles of PCR amplification, using a quantitative real time PCR. The frequency of the mtDNA4977 was significantly higher in the sputum cells of patients with COPD compared to smokers without COPD (p < 0.0001). This difference was not observed in PBLs. Levels of cellular oxidative stress were significantly higher in the sputum cells of subjects with COPD than in the smoker group. However, mtDNA copy number, mitochondrial membrane potential (ΔΨm) and cellular ATP levels in PBLs and sputum cells were not significantly different between the studied groups. The Pearson analysis revealed no correlations between the accumulation of mtDNA4977, and intracellular ATP content and ΔΨm values of the sputum cells, although there was a positive correlation between the increase in the percentage of deleted mtDNA4977 and the levels of cellular oxidative stress in COPD patients (r = 0.80, p < 0.0001). Our studies may suggest that the accumulation of mtDNA4977 in the sputum cells of smokers with COPD does not seem to have an important impact on mitochondrial dysfunction in relation to ATP production and ΔΨm when compared to those of healthy smokers.