Abstract Background Precise and accurate measurements of blood gases are important for the diagnosis, monitoring and treatment of respiratory, metabolic, and acid-base disturbances. This study demonstrates the accuracy of the pH, partial pressure of oxygen (PO2), and partial pressure of carbon dioxide (PCO2) tests in the i-STAT G3+ cartridge with the i-STAT 1 System in arterial, venous, and capillary whole blood specimens in point of care or clinical laboratory settings. Methods The accuracy of the PO2, and PCO2 tests in the i-STAT G3+ cartridge was assessed at external clinical sites in a method comparison study using whole blood specimens across the measurement range for each test: pH 6.5-7.8, PO2 5-700 mmHg, PCO2 5.0-130.0 mmHg. Specimens used in this study were venous, arterial, and capillary specimens prospectively collected under Institutional Review Board-approved informed consent or were leftover de-identified venous or arterial specimens. The study included venous and arterial whole blood samples from ∼140 adult subjects (≥ 18 years) at 3 clinical sites, and capillary whole blood samples from ∼180 adult subject at 3 clinicals sites and ∼25 neonate subjects (birth to 28 days) at 2 clinical sites. The RapidPoint System was used as comparator. For venous and arterial whole blood, samples were collected and tested in duplicate on the i-STAT G3+ cartridge and in singlicate on the RapidPoint System. For capillary whole blood samples, adult fingerstick samples and neonate heel-stick samples were collected in capillary tubes and tested in singlicate on the i-STAT G3+ cartridge and on the RapidPoint System. Passing-Bablok regression was performed for venous and arterial whole blood samples combined, using the first replicate from the i-STAT G3+ cartridge versus the singlicate result from the comparator, and for capillary whole blood samples (adult and neonate combined) using the singlicate result from the i-STAT G3+ cartridge versus the singlicate result from the comparator. Results Passing-Bablok regression of i-STAT G3+ cartridge results versus the comparator was used to calculate slope and correlation coefficient (r). For adult venous and arterial whole blood results, pH had slope = 0.96 and r = 0.99, PO2 had slope = 1.05 and r = 1.00, and PCO2 had slope = 1.04 and r = 0.98. For adult and neonate capillary whole blood results, pH had slope = 1.02 and r = 0.98, PO2 had slope = 1.09 and r = 0.99, and PCO2 had slope = 1.07 and r = 0.96. Conclusions These results demonstrate that the i-STAT 1 System delivers accurate pH, PO2, and PCO2 test results with ∼2 minutes using the i-STAT G3+ cartridge for venous, arterial and capillary whole blood samples for adult and neonate subjects when tested by end-users in a point of care or clinical laboratory setting. This study was funded by Abbott Laboratories.