Abstract The effects of the following factors on acid phosphatase activity in L5178Y cells were studied: inhibitors of RNA, DNA, and protein synthesis, phosphate concentration, growth cycle and cell cycle. Puromycin, high and low phosphate concentrations, and high cell density caused a decrease in enzyme activity while inhibition of DNA synthesis and RNA synthesis caused an increase of activity. During log phase, the cells displayed a constant level of activity which declined to a lower level as the cells entered the stationary phase. In synchronized cells the enzyme activity varied periodically: a minimum in G1 with peaks in late G1-early S and again in late S-G2. Treatment of cells in the log phase with puromycin caused a linear decay of enzyme activity. In synchronized cells, puromycin also caused a linear decay of enzyme activity at all points in the cell cycle and enzyme activity decayed to levels well below those of controls. From this, it is concluded that the cell regulates its level of acid phosphatase activity by varying the rate of synthesis while the rate of decay remains constant throughout the cell cycle.