Abstract. The aim of this study was to evaluate the effect of glucose tolerance test (GTT) on glucose, insulin, non-esterified fatty acids (NEFA) and β-Hydroxybutyrate (BHB) concentration in dairy cows during prepartum and postpartum period. Forty Two clinically healthy Holstein cows were enrolled in two high producing dairy farms in the Northeast Italy. All animals were divided into two equal groups on the basis of the farm of origin: Group A (farm located in Vicenza) and Group B (farm located in Padua). Body condition score (BCS) was determined for both groups. GTT was performed 7 ± 5 days before and 7 ± 5 days after calving. Blood samples were collected from each animal before (T0) and after the administration of glucose at different times. Plasma concentration of glucose, insulin, NEFA and BHB was evaluated. Application of two way analysis of variance (ANOVA) showed no significant effect of farm and a significant effect of transition period (prepartum and postpartum) on BCS for both groups. Multivariate analysis of variance (MANOVA) was applied to determine significant effects of infusion glucose time, transition period and farm on glucose, insulin, NEFA and BHB. No significant difference was observed between Groups A and B and a significant effect of transi tion period and glucose infusion was found on all parameters studied. Results confirm that glucose is an important direct controller of metabolic response in dairy cows during the transition period because of the inappropriate energetic supply that characterizing this physiological phases.