The objectives of this project were to characterize the pharmacodynamics and pharmacokinetics of midazolam in the ball python (Python regius), and to evaluate the effects of midazolam and nitrous oxide (N2O) on the minimum anesthetic concentration of isoflurane (MACIso) in this species. The pharmacodynamics of midazolam were evaluated in a blinded, randomized, crossover study comparing two dosages (1 and 2 mg/kg intramuscular [IM]) in ten ball pythons. There were no significant differences between the two dosages for any parameter evaluated. Midazolam provided moderate sedation and muscle relaxation in all snakes starting 10-15 min post-injection and lasting up to 3-5 days, with a peak effect 60 min post-injection. Heart rates were significantly lower than baseline from 30 min to 5.3 days post-injection with both dosages. In an open trial using 9 ball pythons, the reversal effect of flumazenil was evaluated by administering 0.08 mg/kg IM 60 min after the administration of 1 mg/kg IM of midazolam. Sedation and muscle relaxation were fully reversed within 10 min of administration, but all snakes fully resedated within 3 h. In a semi-blinded, randomized, crossover trial, nine ball pythons were used to evaluate the effects of 1 mg/kg IM midazolam and N2O mixed 50:50 with oxygen on the MACIso. Midazolam significantly decreased the MACIso from 1.11% (95% confidence interval [CI], 0.94-1.28%) to 0.48% (95% CI, 0.29-0.67%), and N2O decreased the MACIso to 0.92% (95% CI, 0.74-1.09%). No clinically relevant differences between treatments were noted regarding blood gases but isoflurane induction with face mask caused marked acidemia that resolved with mechanical ventilation. Central venous catheters were surgically placed in twelve ball pythons to evaluate the pharmacokinetics of midazolam. Snakes were randomly and equally divided into two groups receiving 1 mg/kg of midazolam either intracardiac (IC; group 1) or IM (group 2). Plasma concentrations of midazolam and its major metabolite 1-hydroxymidazolam were determined using high-performance liquid chromatography tandem-mass spectrometry. Mean bioavailability following IM administration was 89%. Mean ± SD clearance of midazolam was 0.053 ± 0.008 L/h/kg, leading to slow elimination half-lives (12.04 ± 3.25 h [IC] and 16.54 ± 7.10 h [IM]).