Despite their relevance for neuronal Ca²⁺-induced Ca release (CICR), activation by Ca²⁺ of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg²⁺] and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis (cytoplasmic) free [ATP], [Mg²⁺] and RyR redox state on the Ca²⁺ dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylyl-imidodiphosphate (AMP-PNP), increasing free [Mg²⁺] up to 1 mM inhibited vesicular [³H]-ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg²⁺ inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca²⁺ dependencies, defined by low, moderate or high maximal fractional open time (P_o), which depend on RyR redox state as we have previously reported. In all cases, cis ATP addition (3 mM) decreased threshold [Ca²⁺] for activation, increased maximal P_o and shifted channel inhibition to higher [Ca²⁺]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis [Mg²⁺] up to 1 mM inhibited low activity channels > moderate activity channels, but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg²⁺] induced a right-shift in Ca²⁺ dependence for all channels so that [Ca²⁺] < 30 µM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca²⁺ at physiological [ATP] and [Mg²⁺]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR.