The objective was to determine peripheral activity of COMT in pregnant versus non-pregnant lactating Holstein cows. Cows (n = 22) were synchronized using the Ovsynch plus CIDR protocol and bred via artificial insemination on d 0. Cows were retrospectively classified as pregnant (n = 4) or open (n = 14) based on rectal palpation at d 35 post-insemination. Moreover, cows were classified as rebred (n = 4) if they showed signs of estrus and were re-inseminated before rectal palpation. Blood samples were collected on d 0, 4, 8, 12, 16, 20, and 24 post-insemination. Plasma was separated by layering 3 mL of blood onto 3 mL of Ficoll and centrifuged at 400 × g at room temperature for 30 min. One mL of erythrocytes were collected and stored at −80°C. Peripheral activity of COMT was determined by incubating erythrocyte cell homogenates with 2 mM s-adenosyl methionine, 3 mM cysteine, 10 mM MgCl₂, and 1 mM of the COMT specific substrate 6–7-dihydroxycoumarin. Samples and substrates were incubated at 37°C for 30 min and enzyme reactions were stopped by adding 0.5 M HCl, 10% NaNO₂, 10% NaMoO₄, and 1 M NaOH to each sample. Lastly, the disappearance of 6–7-dihydroxycoumarin was determined by measuring the amount of light absorbed at 510 nm using a Spectra Max Plus plate reader. Activity of COMT was analyzed using repeated measures ANOVA of the MIXED procedure of SAS and the model statement included day, pregnancy status, and their respective interaction. Activity of COMT was increased (P < 0.01) on d 16 post-insemination compared with all other days in cows classified as open or pregnant. Activity of COMT was not different (P = 0.87) throughout the sampling period in cows classified as rebred. Moreover, activity of COMT was increased (P < 0.01) on d 16 in pregnant cows versus rebred cows. Therefore, peripheral activity of COMT in lactating Holstein cows, which is involved in catechol-estrogen metabolism, may be altered by pregnancy status as well as days post-insemination.