The effects of Mg²⁺ and Ba²⁺ on single-channel properties of the inositol 1,4,5-trisphosphate receptor (IP₃R) were studied by patch clamp of isolated nuclei from Xenopus oocytes. In 140 mM K⁺ the IP₃R channel kinetics and presence of conductance substates were similar over a range (0-9.5 mM) of free Mg²⁺. In 0 mM Mg²⁺ the channel current-voltage (I-V) relation was linear with conductance of ~320 pS. Conductance varied slowly and continuously over a wide range (SD  60 pS) and sometimes fluctuated during single openings. The presence of Mg²⁺ on either or both sides of the channel reduced the current (blocking constant ~0.6 mM in symmetrical Mg²⁺), as well as the range of conductances observed, and made the I-V relation nonlinear (slope conductance ~120 pS near 0 mV and ~360 pS at ±70 mV in symmetrical 2.5 mM Mg²⁺). Ba²⁺ exhibited similar effects on channel conductance. Mg²⁺ and Ba²⁺ permeated the channel with a ratio of permeability of Ba²⁺ to Mg²⁺ to K⁺ of 3.5:2.6:1. These results indicate that divalent cations induce nonlinearity in the I-V relation and reduce current by a mechanism involving permeation block of the IP₃R due to strong binding to site(s) in the conduction pathway. Furthermore, stabilization of conductance by divalent cations reveals a novel interaction between the cations and the IP₃R.