Serum alkaline phosphatase (ALP) activities were measured during dry and lactational periods to investigate the influence of lactation on serum ALP activity in cows. Higher levels of serum ALP activity were seen in lactational periods than in dry periods. The serum activities of bone-specific ALP (BALP), liver ALP (LALP), tartrate resistant acid phosphatase (TRAP) and aspartate aminotransferase also increased in lactational periods. ALP activities in the bone extract and in whey were decreased at similar rates by the addition of lectin. Moreover, since the ALP band in whey was observed to have the same migration in polyacrylamide gel (PAG) disk electrophoresis as that of the bone extract, analysis of ALP isoenzymes by lectin affinity or PAG disk electrophoresis could not distinguish ALP originating from the mammary gland from that of bone. In this study, it was clear that the increased level of serum ALP activity was due to increases of BALP and LALP in lactational periods. However, the extent of the influence of ALP originating from the mammary glands on serum ALP activity was unknown. Judging from changes of BALP and TRAP activities in the serum and the correlation between the both, it was guessed that ALP originating from the mammary glands influenced serum ALP activity.