To characterize the relationship of changes in diaphragmatic thickness during contraction to changes in lung volume, we developed a technique to measure diaphragm thickness based on M-mode ultrasonography. First, diaphragmatic thickness was measured in situ at necropsy with ultrasound and verified by measuring the same resected segment of diaphragm by ruler (correlation coefficient = 0.93, slope = 0.97). The technique of imaging the diaphragm in living subjects was developed by using a 15-MHz transducer coupled to an M-mode echocardiograph. Ten normal male volunteers were studied while sitting. The ultrasound transducer was held between the ribs in the ninth lateral interspace, and tidal volume was measured by spirometry. The thickening fraction (TF) was calculated as TF = (thickness at peak inspiration - thickness at end expiration)/thickness at end expiration for each of a series of different sized breaths. The function, TF vs. lung volume, for a range of volumes was linear for each subject and had intrasubject reproducibility with intersubject variability. We conclude that diaphragmatic TF is related to function as determined by lung volume, and this may prove to be a useful technique for in vivo studies of diaphragmatic function.