Mitochondria are central to metabolism and the primary energy producers for all biosynthesis, including lactation. The objective of this study was to determine if high and low producing dairy cows exhibit differences in mitochondrial enzyme activities during early lactation. Fifty-six Holstein cows were assigned to one of 4 groups: primiparous high or low ECM (41.8 vs 33.2 kg/d) and multiparous high or low ECM (56.8 vs 44.3 kg/d). Group assignments were made after data were collected by averaging ECM for primiparous cows and then for multiparous cows and assigning below average cows to the low group and above average cows to the high group. Whole blood samples were collected at one time point within (70 ± 11 DIM) and processed for crude mitochondrial extracts from peripheral blood mononuclear cells (PBMCs). Mitochondrial function of the extracts was assessed by measuring the activity rates of citrate synthase, complex I, complex IV, and complex V using kits from Abcam (Cambridge, MA). Milk samples were collected 9 times within a week of blood collection and analyzed for major components using a MilkoScan FT2 by FOSS (Mulgrave, Australia). Data were analyzed using the Mixed procedure of SAS (Version 9.4, SAS Inst. Inc., Cary, NC) for high and low ECM with cow as the experimental unit of interest and dependent variables parity, and DIM as a covariate. Complex V enzyme activities tended to be higher in multiparous than primiparous cows (0.30 vs. 0.20 mOD/min/ug) respectively; (P = 0.1). Citrate synthase activity was lower in multiparous animals than primiparous (0.92 vs.1.3 mOD/min/ug) respectively; (P = 0.006). Complex I activity was lower in low producing cows than high producing cows for both primiparous and multiparous groups (0.022 vs. 0.020 mOD/min/ug), (0.028 vs. 0.013 mOD/min/ug), respectively; (P = 0.04) and not affected by parity (P > 0.1). These findings suggest that complex I enzyme activity may be a marker of ability to produce milk and support previous findings that mitochondrial density (citrate synthase activity) decreases with age in dairy cattle.