Abstract OBJECTIVE To evaluate the efficacy of tiletamine-zolazepam (TZ) for sedation of red-eared sliders and to assess the effect of injection site location on sedation parameters. METHODS Over a 1-month trial period, healthy turtles obtained from a rescue were administered TZ (10 mg/kg, SC) in the forelimb (FL) or hind limb (HL) in a randomized, blinded crossover design in a laboratory setting. Heart rate; respiratory rate; neck, forelimb, and hind limb muscle tone; pelvic limb withdrawal reflex; jaw tone; cloacal tone; and feasibility of positioning for jugular venipuncture were evaluated. Flumazenil at 0.05 mg/kg was administered SC 45 minutes after TZ injection for recovery. Time to first effects, total sedation score, and time to recovery were recorded. RESULTS There was no significant difference in mean time to initial sedative effects between FL (11 ± 4 minutes) and HL (14 ± 6 minutes) treatments in the study population (n = 8 turtles). Six of 8 turtles (75%) and 5 of 8 turtles (63%) in the FL and HL treatments, respectively, developed decreased muscle tone. Simulated jugular venipuncture was successful in 7 of 8 turtles (88%) in both treatments, although additional restraint was required in 3 of 8 turtles (43%) in the HL group. There was no significant difference in total sedation score at any time point between treatments, and median heart rate across the sedative period was similar between treatment groups. Mean time to recovery was similar between treatments (FL, 135 ± 55 minutes; HL, 165 ± 45 minutes). CONCLUSIONS Tiletamine-zolazepam (10 mg/kg) produced moderate sedation of red-eared sliders. The location of SC TZ injection administration did not significantly affect sedation parameters in this species. CLINICAL RELEVANCE Subcutaneous TZ can be used for sedation of red-eared sliders in clinical practice to facilitate examination and blood collection in this species.