This study investigated changes in serum levels of hepatic, bone, and intestinal alkaline phosphatase (ALP) isoenzymes (ALP2, ALP3, and ALP5, respectively) in Holstein cows around parturition. Tartrate-resistant acid phosphatase 5b (TRAP5b) activity and calcium (Ca) concen- trations were also measured. We analyzed blood samples from 11 late-pregnant heifers (primipa- rous group) and 13 multiparous (2-4 lactations; multiparous group) cows at 3 weeks (18-24 days prepartum; -3 weeks), 2 weeks (17-11 days prepartum; -2 weeks), and 1 week (10-4 days prepar- tum; -1 weeks) before parturition; the day of calving (within 12 h post-calving; day 0); and 5 days postpartum (5 days). ALP3 activity was significantly higher in the primiparous group than in the multiparous group, whereas the activities decreased significantly in both groups after 5 days. ALP2 and ALP5 activities did not change, whereas ALP2 activity was significantly higher in the primiparous group than in the multiparous group. TRAP5b activity was significantly higher in the primiparous group than in the multiparous group and showed a transient significant increase at day 0. Ca concentration significantly decreased at day 0 in both groups; the Ca level at day 0 was significantly higher in the primiparous group than in the multiparous group. These data show that ALP3 activity in serum may indicate a change in osteoblastic bone forma- tion after calving, but further study is needed to determine the clinical application for measuring ALP isoenzymes in bovine medicine.