The chemotherapeutic anthracycline metabolite doxorubicinol (doxOL) has been shown to interact with and disrupt the function of the cardiac ryanodine receptor Ca21 release channel (RyR2) in the sarcoplasmic reticulum (SR) membrane and the SR Ca21 binding protein calsequestrin 2 (CSQ2). Normal increases in RyR2 activity in response to increasing diastolic SR [Ca21] are influenced by CSQ2 and are disrupted in arrhythmic conditions. Therefore, we explored the action of doxOL on RyR2’s response to changes in luminal [Ca21] seen during diastole. DoxOL abolished the increase in RyR2 activity when luminal Ca21 was increased from 0.1 to 1.5 mM. This was not due to RyR2 oxidation, but depended entirely on the presence of CSQ2 in the RyR2 complex. DoxOL binding to CSQ2 reduced both the Ca21 binding capacity of CSQ2 (by 48%–58%) and its aggregation, and lowered CSQ2 association with the RyR2 complex by 67%–77%. Each of these effects on CSQ2, and the lost RyR2 response to changes in luminal [Ca21], was duplicated by exposing native RyR2 channels to subphysio-logic (#1.0 mM) luminal [Ca21]. We suggest that doxOL and low luminal Ca21 both disrupt the CSQ2 polymer, and that the association of the monomeric protein with the RyR2 complex shifts the increase in RyR2 activity with increasing luminal [Ca21] away from the physiologic [Ca21] range. Subsequently, these changes may render the channel insensitive to changes of luminal Ca21 that occur through the cardiac cycle. The altered interactions between CSQ2, triadin, and/or junctin and RyR2 may produce an arrhythmogenic substrate in anthracycline-induced cardiotoxicity.