Although carboxyhemoglobin (COHb) is often increased in smokers, American Thoracic Society recommendations for adjusting the single breath carbon monoxide (CO) diffusing capacity (DL(CO)(SB)) for COHb remain optional. Using a previously described 3-equation technique, we measured DL(CO)(SB) and an index of diffusion inhomogeneity (DI) in 10 healthy, nonsmoking subjects who performed DL(CO)(SB) maneuvers both before and after increasing COHb. CO backpressure (FA(CO)) was measured from the exhaled gas of a standardized deep breath of room air that immediately preceded each DL(CO)(SB) and was validated by measurement of FA(CO) from an identical "sham" maneuver without inhaling CO. Without adjustments for FA(CO) or COHb, DL(CO)(SB) decreased with increasing COHb. This effect persisted when DL(CO)(SB) was adjusted only for FA(CO), but it was eliminated with further adjustment for the anemia effect of increasing COHb. The anemia adjustment was proportional to the fractional COHb. DI, adjusted for FA(CO), was unaffected by increasing COHb. We conclude that DL(CO)(SB) must be adjusted for both the buildup of CO backpressure and the anemia effect of increasing COHb. Adequate corrections of DL(CO)(SB) can be implemented using FA(CO) measured during a standardized deep breath immediately preceding the DL(CO)(SB) maneuver. Current American Thoracic Society recommendations for DL(CO)(SB) standardization do not adequately compensate for COHb.