The murine S49 lymphoma cell transports Mg2+ by a system distinct from systems responsible for Ca2+ influx (J. Physiol. London 337: 351-371, 1983). We have now determined the ability of various cations, anions, and drugs to modulate Mg2+ influx. Neither sulfate, nitrate, phosphate, nor bicarbonate altered Mg2+ influx. Among cations only T1+, Ba2+, Zn2+, Mn2+, Sc3+, and La3+ potently inhibited Mg2+ influx without causing obvious cell toxicity. Seventeen other cations were ineffective at maximal nontoxic concentrations. T1+ inhibition (Ki = 300 micron) is noncompetitive and apparently derives from its ability to dissipate membrane potential. The noncompetitive nature of and the rather poor inhibition constants for Ca2+ (Ki approximately equal to 5 mM) and Mn2+ (Ki = 200 micron) indicate that neither cation is an effective physiological antagonist of Mg2+ influx. Only Ba2+ exhibited competitive inhibition of Mg2+ influx (Ki = 1 mM). Cisplatin and Ca2+ channel antagonists also did not inhibit Mg2+ influx. These data further differentiate Mg2+ transport systems from those for Ca2+. In addition, the selectivity series for group IIa cation inhibition of influx (Mg2+ greater than Ba2+ much greater than Ca2+ greater than or equal to Sr2+) has not been observed previously in biological systems and is indicative of a very high anionic field strength at the Mg2+ recognition site.