Evaluation of blood gas parameters is a suggested method for assessing test article effects on respiratory function in nonclinical safety pharmacology and toxicology studies. The sensitivity of the ABL80 FLEX CO-OX instrument to measure arterial blood gas measurements has not been thoroughly evaluated in rats. This study evaluated intra-precision, inter-precision, stability, intra-individual variability, and type of collection apparatus for blood gas collections. Additionally, this study com-pared blood gas values to head-out plethysmography data in Sprague Dawley rats administered a respiratory stimulant; 20 mg/kg theophyl-line via oral gavage. Blood was collected from the femoral artery via arterial catheters and analyzed with the ABL80 FLEX CO-OX blood gas analyzer using Radiometer assays. Intra-precision and inter-precision variability was acceptable for all endpoints, with the exception of standard base excess, actual base excess, and anion gap (K+). The following endpoints exhibited acceptable room temperature stability through 30 minutes post-collection: bicarbonate, concentrations of hemoglobin, sodium ions and chloride ions, fractions of methemoglobin and carboxyhemoglobin. Although results from Pico syringes, Westmed (Pico 3100-25) syringes, and capillary samples were generally similar, it is recommended that one type of collection device be used within a study, rather than using these devices interchangeably. In head-out plethysmography evaluation, theophylline significantly increased tidal volume, respiration rate, and minute volume within 2 hours postdose. However, fractions of carboxyhemoglobin and methemoglobin and concentrations of bicarbonate, calcium ions and hemoglobin were minimally decreased by theophylline; other blood gas parameters were not significantly altered during this same postdose time span. These data suggest that blood gas measurements are less sensitive than plethysmography for nonclinical safety evaluation of respiratory safety pharmacology, but blood gas parameters can be valuable supplemental data.