The pharmacokinetics of dantrolene and its active metabolite, 5-hydroxydantrolene, after a single oral dose of either 5 or 10 mg/kg of dantrolene was determined. The effects of exposure to dantrolene and 5-hydroxydantrolene on activated whole-blood gene expression of the cytokines interleukin-2 (IL-2) and interferon-γ (IFN-γ) were also investigated. When dantrolene was administered at a 5 mg/kg dose, peak plasma concentration (Cmax) was 0.43 μg/mL, terminal half-life (t1/2) was 1.26 h, and area under the time–concentration curve (AUC) was 3.87 μg·h/mL. For the 10 mg/kg dose, Cmax was 0.65 μg/mL, t1/2 was 1.21 h, and AUC was 5.94 μg·h/mL. For all calculated parameters, however, there were large standard deviations and wide ranges noted between and within individual dogs: t1/2, for example, ranged from 0.43 to 6.93 h, Cmax ratios ranged from 1.05 to 3.39, and relative bioavailability (rF) values ranged from 0.02 to 1.56. While activated whole-blood expression of IL-2 and IFN-γ as measured by qRT-PCR was markedly suppressed following exposure to very high concentrations (30 and 50 μg/mL, respectively) of both dantrolene and 5-hydroxydantrolene, biologically and therapeutically relevant suppression of cytokine expression did not occur at the much lower drug concentrations achieved with oral dantrolene dosing.