Temporal and spatial profiles of alkaline phosphatase (ALP) activity during the development of amphioxus have not been fully documented and thus the present study investigated this activity spectrophotometrically, electrophoretically and histochemically. The following results were observed: (1) spectrophotometrically, ALP activity increased markedly at the late gastrula stage and reached a plateau at 15 hr postfertilization; (2) the electrophoretic pattern of ALP isozymes changed dramatically during development; (3) ALP activity was initially localized in the posterior wall of the primitive gut and the anterior 5 to 6 somites at about the 15 hr larva stage, and then in the notochord and all the somites at about the 18 hr larva stage; (4) in 1-day larvae, ALP activity decreased in the posterior wall of the primitive gut and in the anterior 5 to 6 somites which had ALP activity at 15 hr, but it appeared in the newly formed somites, especially in the myosepta, the crevices cut in between adjoining somites; (5) in 2-day larvae, ALP activity was no longer visible in somites but became highly evident in most of the notochord except for its rostral region; and (6) when the lateral plate mesoderm pushed down ventrally on either side of the intestine and conjoined beneath the intestine, ALP activity was also detected in the conjoining lateral plate mesoderm. Apparently, two types of ALP exist in amphioxus larvae, the transient endodermal ALP and the constant mesodermal ALP, and the spatial and temporal correlation of ALP activity with the developing mesoderm, including the notochord, suggests that it plays a role in the differentiation of mesodermal structures during the development of amphioxus.