Carbon monoxide diffusing capacity (DLCO) or transfer factor (TLCO) is a particularly useful test of the appropriateness of gas exchange across the lung alveolocapillary membrane. With the purpose of establishing predictive equations for DLCO using a non-smoking sample of the adult Brazilian population, we prospectively evaluated 100 subjects (50 males and 50 females aged 20 to 80 years), randomly selected from more than 8,000 individuals. Gender-specific linear prediction equations were developed by multiple regression analysis with single breath (SB) absolute and volume-corrected (VA) DLCO values as dependent variables. In the prediction equations, age (years) and height (cm) had opposite effects on DLCOSB (ml min-1 mmHg-1), independent of gender (-0.13 (age) + 0.32 (height) - 13.07 in males and -0.075 (age) + 0.18 (height) + 0.20 in females). On the other hand, height had a positive effect on DLCOSB but a negative one on DLCOSB/ VA (P < 0.01). We found that the predictive values from the most cited studies using predominantly Caucasian samples were significantly different from the actually measured values (P < 0.05). Furthermore, oxygen uptake at maximal exercise (VO2max) correlated highly to DLCOSB (R = 0.71, P < 0.001); this variable, however, did not maintain an independent role to explain the VO2max variability in the multiple regression analysis (P > 0.05). Our results therefore provide an original frame of reference for either DLCOSB or DLCOSB/VA in Brazilian males and females aged 20 to 80 years, obtained from the standardized single-breath technique.