The lung diffusion capacity (DLCO) determined by the single-breath technique greatly helps in the differential diagnosis and classification of severity of common lung diseases. However, widespread use of single-breath DLCO tests in Latin America has been limited, in part, by the lack of appropriate reference values. Our objective was to derive robust reference equations for single-breath DLCO from healthy Hispanic adults, using the most recent guidelines and taking into account altitude above sea level and hemoglobin.We recruited healthy adults from Caracas (690 m), Santiago (650 m), Mexico City (2,240 m), and Bogota (2,640 m). DLCO testing was completed using an instrument that exceeds American Thoracic Society/European Respiratory Society 2005 guidelines for spirometry and single-breath DLCO and provided centralized training and a quality assurance program.We included 480 healthy Hispanic adults (58.3% women) with a mean age of 46 y (range 22-83 y). Their mean ± SD single-breath DLCO was 30.4 ± 9.2 mL/min/mm Hg. Results as a percentage of predicted by Crapo's reference values (the closest to obtained values) were 83 ± 10% (Caracas), 91 ± 10% (Santiago), 104 ± 17% (Mexico City), and 118 ± 19% (Bogota), and current suggested adjustments by hemoglobin or altitude did not correct differences, especially in Santiago and Caracas.We recommend these new single-breath DLCO reference equations to predict single-breath DLCO in Latin America performed with current instruments and procedures and including as a predictor altitude above sea level.