The study objective was to evaluate the sedative, muscle relaxant, and cardiorespiratory effects of midazolam and flumazenil in the ball python (Python regius). Ten healthy adult female ball pythons were used in a randomized and blinded crossover trial evaluating the effects of two dosages (1 and 2 mg/kg intramuscular [i.m.] in the cranial third of the body). In a subsequent open trial, nine ball pythons received 1 mg/kg i.m. of midazolam followed by 0.08 mg/kg i.m. of flumazenil 60 min later. Heart rate, respiratory rate, temperature, and the level of sedation and muscle relaxation (using a semiobjective scoring system) were evaluated. There were no significant differences between midazolam dosages for any of the parameters evaluated. Sedation scores were significantly increased compared with baseline from 15 min (1 mg/kg) and 10 min (2 mg/kg) postinjection up until 56 hr (1 mg/kg) and 72 hr (2 mg/kg) postinjection. Peak effect was reached 60 min postinjection, with 60% of snakes (6/10) being unable to right themselves. One snake developed paradoxical excitation with the 2 mg/kg dosage. Heart rates were significantly lower than baseline from 30 min to 128 hr postinjection with both midazolam dosages. Respiratory rates were significantly lower than baseline at four time points, with the highest dosage only: 15, 45, 60 min, and 8 hr postinjection. Flumazenil resulted in reversal of sedation and muscle relaxation in all snakes within 10 min of administration. However, resedation was evident in all snakes 3 hr after reversal. Midazolam administered at 1 and 2 mg/kg i.m. provides a moderate to profound, although prolonged, sedation and muscle relaxation in ball pythons. Flumazenil reverses the effects of midazolam in ball pythons, but its duration of action at the evaluated dosage is much shorter than midazolam, leading to resedation.