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Rates of alcohol use and alcohol-related harms increase with greater alcohol availability. However, regional differences in sociodemographic characteristics and built environment may affect this association. This study evaluated the association between off-premise alcohol availability and alcohol use in Ontario, Canada, and the degree to which this association varies between cities.This was a cross-sectional spatial analysis of urban neighborhoods in Ontario, Canada (There was an overall positive association between alcohol availability and high-volume alcohol use (male coefficient estimate (β) = 0.19, 95% credible interval [CI] [0.16, 0.22]; female β = 0.17, 95% CI [0.13, 0.21]). However, the association was eliminated in models that allowed for this association to vary between cities via an interaction term (male β = -0.04, 95% CI [-0.26, 0.19]; female β = -0.04, 95% CI [-0.34, 0.26]). This was explained by variability in the association between cities, where some cities demonstrated a positive association between availability and use and others demonstrated a negative association.Although there is a province-wide positive association between off-premise alcohol availability and high-volume alcohol use, there is substantial regional variation in this association that may affect the local effectiveness of alcohol regulation policies.