Abstract. Transition dairy cows often enter a stage of negative energy balance during which the utilization of energy reserves is reflected in the milk fatty acid (FA) composition. In this study, metabolic status was evaluated by measuring milk FA, ruminal short-chain FA (RSCFA), and serum biochemical parameters in Holstein cows. Samples (milk, rumen contents, and blood) were collected around days 30 (early) and 150 (middle) of lactation, and rumen contents and blood samples were collected 30 days before calving (dry). Fatty acids were extracted and FA composition was determined. Glucose, triacylglycerols, total cholesterol, low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), beta-hydroxybutyric acid (BHB), and non-esterified fatty acid (NEFA) concentrations were determined in serum samples. Lower percentages of saturated FA in milk and higher percentages of monounsaturated FA, polyunsaturated FA, and C18:1n-9 were observed in early lactation compared to mid-lactation. In rumen higher concentrations of propionic acid were determined at mid-lactation compared to early lactation. Acetic and butyric acid concentrations showed no significant differences between sampling intervals. In serum higher glucose concentrations were observed during the dry period and mid-lactation than during early lactation. Lower BHB and higher NEFA concentrations were noted during early lactation compared to mid-lactation and the dry period. Total cholesterol, LDL-C, HDL-C and triacylglycerols showed no significant differences between sampling intervals. The results of the present study suggest that determination of milk FA is a potential indicator of energy status in dairy cows.