In this study we determined reference values of total diffusing capacity of carbon monoxide (DLCO) and DLCO per liter alveolar volume (DLCO/VA) at total lung capacity (TLC) and at lung volumes below TLC in sitting position. In 55 healthy nonsmoking volunteers (20-85 yr old), we determined reference values at TLC level in which age was the only parameter. In a subgroup (n = 16) these references did not change by correction for normal variability in hemoglobin concentration. In all volunteers DLCO decreased and DLCO/VA increased with decreasing VA. The increase in DLCO/VA was linear and less in older subjects. We derived equations to calculate reference values of DLCO/VA for lung volumes at and below TLC with two methods: 1) "random coefficients linear" model, which calculates the reference values directly, and 2) a conversion method, which calculates DLCO/VA for lower VA levels from reference values at TLC. An advantage of the conversion method is the suitability of DLCO/VA reference values at TLC of other populations. A disadvantage is the greater standard deviation of these reference values compared with those obtained by the random coefficients linear method. DLCO can be found by multiplying DLCO/VA with VA.