Amphioxus, a cephalochordate, is an important model fish for studies in evolution and comparative biology. A successful cell culture from amphioxus tissues in vitro would help understanding some basic issues. To determine the optimal culture conditions for proliferation of amphioxus cells, primary cultures were initiated from buccal cirri, tail, gill, gut and metapleural fold of amphioxus Branchiostoma belcheri tsingtauense. The media tested were L-15, F-12, M 199, MEM, DMEM, PRMI 1640 and LDF, each was supplemented with 20% fetal bovine serum. The optimal conditions include tail tissue cultured in L-15 or F-12 with supplement of 20% FBS and 1.5% NaCl at about 25°C.