We and others frequently have noted serum potassium levels of 8.0 ± 0.85 mEq/L or greater in laboratory mice; this concentration has even been published as the upper limit of a 'normal' reference range. However, if bone fide, this potassium concentration would be incompatible with life in all species. We investigated conditions frequently encountered in the research setting to distinguish artifactual from true hyperkalemia. Variables evaluated included site of collection, time allowed for clot formation before serum separation, time elapsed between collection and analysis of samples collected in a serum separator tube, precollection method of anesthesia, and euthanasia technique. Serum potassium was measured from 75 C57BL/6NTac 10-wk-old female mice and divided into at least 5 mice per variable. Animals were euthanized by exsanguination immediately after terminal CO2 or ketamine–xylazine (KX) administration. Mice euthanized with CO2 had higher mean serum potassium (7.0 ± 0.5 mEq/L) and range serum potassium (6.0 to 8.1 mEq/L) than did KX-treated mice. CO2 inhalation resulted in significantly lower blood pH (6.9 ± 0.1), higher pCO2 (153.3 ± 38.8 mm Hg), and higher lactate levels (3.9 ± 0.9 mmol/L) than did KX anesthesia followed by exsanguination. These results suggest that antemortem respiratory acidosis from CO2 administration causes artifactual hyperkalemia in mice. Therefore, blood collection under KX anesthesia is preferable over CO2 inhalation to obtain accurate potassium values from mice.