We measured the bone-specific alkaline phosphatase (ALP) isoenzyme activity in 67 plasma samples from 14 newborn Holstein calves using both a conventional method (featuring heat inactivation) and a commercial agarose gel electrophoresis (AGE) kit; the relevant isoenzymes were termed bone-specific ALP (BAP) and ALP isoenzyme 3 (ALP3). We explored whether the AGE kit afforded reliable data when used to analyze samples from Holstein calves. The blood was collected from the jugular vein of each calf immediately prior to the first colostrum feeding (pre-feeding), 20 and 40 h after pre-feeding, and on days 4 and 7; whereas three samples (from three calves) were not obtained. The total plasma ALP activity varied widely, exceeding the ranges of reference values. On electrophoresis, 52 of 67 plasma samples (77.6 %) clearly contained both ALP isoenzyme 2 and ALP3, as did control human serum. The total ALP activity of the 52 samples ranged from 166–1989 U/L (median: 1013 U/L), whereas the values for the other 15 samples (22.4%) exhibiting abnormal isoenzyme fractionation ranged from 1014–5118 U/L (median: 1780 U/L). In the 52 plasma samples exhibiting clearly separated isoenzymes, ALP3 and BAP activities were strongly positively correlated as revealed by Deming regression (y = 0.93x + 22.6, p<0.0001) and Bland-Altman analysis (ALP3/BAP activities limit of agreement: −5.1%). Thus, the AGE kit yields useful information on newborn calves, and can replace the conventional method when the total plasma ALP activity is less than approximately 1000 U/L.