Determination of ¹³CO₂ enrichment on the CO₂ released from blood by acid has been used in situations in which breath sampling is difficult. This method can be improved by measuring this enrichment on the CO₂ spontaneously released from blood. Therefore, simultaneous comparisons of ¹³CO₂ content between breath and arterialized blood added with or without acid were performed in 51 samples from human studies, using the statistical method of Bland and Altman (J. M. Bland and D. G. Altman. Lancet 1: 307-310, 1986). Strong relationships exist between the methods (r > 0.99) expressed in atom percent excess (APE). Compared with breath, the acid method overestimates the ¹³CO₂ enrichment (0.318 ± 0.632 APE × 1,000, P < 0.001). The acid-free method shows similar enrichments to breath (0.003 ± 0.522 APE × 1,000, P = 0.97) with good precision and degree of agreement (95% confidence interval 0.15 APE × 1,000). The analysis can be performed up to 5 days after sampling with a good reproducibility. In conclusion, measuring ¹³CO₂ enrichments on the CO₂ spontaneously released from blood is feasible, gives identical results to the breath method, and lessens operator manipulations. It allows study of situations in which the breath sampling method is not feasible.