This paper reports a procedure used for isolating the entire epithelial lining of the rat trachea. Isolated trachea was initially filled with 0.2% hyaluronidase and incubated at 37 degrees C for 30 min. Tracheas were flushed with medium and then reinflated with 0.5 microgram/ml cytochalasin B and re-incubated for 60 min. The tracheal lumens were again flushed and reinstilled with 24 iu/ml pronase and incubated for a further 30 min. The tracheas were flushed again and the cells removed enumerated and viability assessed by trypan blue dye exclusion. Cell yields (X 10(6)) from 30 consecutive Fischer 344 rats were 5.06 +/- 0.16 (s.e.m.) and the mean percentage of viable cells was 83.13 +/- 1.10 (s.e.m.). This cell yield was close to the estimated tracheal cell population (5.3 X 10(6)). The suspensions were predominantly single cells which apparently retained a normal ultrastructural appearance.