It is shown that Streptococcus pneumoniae possesses a Ca2+ transporter, sensitive to the amiloride derivative 2',4'-dimethylbenzamil (DMB), which is essential for grown at high Ca(2+)-concentrations, and which mediates the triggering by Ca2+ of competence for genetic transformation in the exponential phase and autolysis in the late exponential phase. DMB inhibited both Ca2+ transport and the Ca2+ response. Kinetic analysis of 45Ca2+ transport in ATP-depleted S. pneumoniae revealed an electrogenic influx sensitive to DMB. This transport was cooperative with respect to Ca2+ concentration, and exhibited a Hill coefficient (nH) of 2. In bacteria pre-loaded with 45Ca2+, a DMB-sensitive efflux could be triggered by an imposed Na+ gradient. The efflux kinetics showed the same cooperativity profile as Ca2+ concentration and a similar nH value to that of influx, suggesting a possible Na+/Ca2+ antiport. Cooperativity of transport was lowered (nH = 1) by a mutation that confers resistance to DMB and abolishes the Ca2+ response. These results demonstrate that DMB-sensitive Ca2+ transport is essential for growth and competence regulation. The role of the DMB-sensitive porter involved in Ca2+ circulation and in Ca2+ homeostasis and its possible regulation by competence factor are discussed.