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Streptozotocin (STZ) is the most used diabetogenic chemical for creating rat models of type 1 and type 2 diabetes. Despite~ 60 years of using STZ in animal diabetes research, some prevailing views about STZ preparation and use are not supported by evidence. Here, we provide practical guides for using STZ to induce diabetes in rats. Susceptibility to the diabetogenic effect of STZ is inversely related to age, and males are more susceptible to STZ than females. Wistar and Sprague-Dawley rats, the most commonly-used rat strains, are sensitive to STZ, but some strains (eg, Wistar-Kyoto rats) are less sensitive. STZ is mostly injected intravenously or intraperitoneally, but its intravenous injection produces more stable hyperglycemia. Despite the prevailing view, no fasting is necessary before STZ injection, and injection of its anomer-equilibrated solutions (ie, more than 2 hours of dissolving) is recommended. Mortality …