The effects of Mg2+ on single mechanosensitive (MS) channel currents recorded from Xenopus oocytes were studied using cell-attached and inside-out patch configurations. Mg2+ both permeates and blocks MS channels. Under symmetrical ionic conditions, the blocking effects of Mg2+ can be described by a Hill coefficient of 0.9 at +/-100 mV and IC50s of 0.12 mM (-100 mV) and 0.60 mM at (+100 mV). Although block by intracellular Mg2+ may contribute to inward MS channel rectification, significant current rectification is retained even under symmetrical KCl concentrations and in the complete absence of Mg2+. The observed voltage dependencies of the IC50 for Mg2+ block and the Km for K+ current saturation indicate asymmetries in the MS channel pore. In addition, the absence of K+ self block and anomalous mole fraction effects with K+/Tl+ mixtures indicate a single site pore model.