In the course of serial passages for several years a line of uncloned HeLa cells (A line) showed a gradual decrease in plaquing efficiency by coxsackievirus A9 (CA9 virus), while subcultures prepared from the same line kept frozen at an early passage level (A original line) did not show any change. However, it was observed later that the plaque-forming ability of the A original line (A orig. line) also decreased after serial passages as was observed with the A line. Comparing the characteristics of the same cell line at two different passage levels, it was found that the efficiency of adsorption of virus to cells was nearly the same, while virus yield at 8 hours after infection was different. The activity of alkaline phosphatase of cells was also different between these two passage levels, suggesting that a high enzymatic activity is associated with the susceptibility of cell cultures to CA9 virus. Magnesium chloride at 25 mM enhanced plaque formation by CA9 virus in highly passaged less susceptible cell cultures, and a possible role of the chemical as a stabilizer of alkaline phosphatase was discussed.