This study aimed to establish whether the bacterial density of spontaneous sputum is affected by the time and mode of sample storage. Ten patients with bronchiectasis collected all sputum expectorated over 45 min. The samples were aliquoted and processed at 25 degrees C for qualitative and quantitative bacteriology at 1, 2, 4 and 6 h from expectoration. Further aliquots were stored at 25 degrees C, 4 degrees C and -20 degrees C for 24 and 48 h prior to processing. The species present was identified and median (interquartile range) sputum log(10) bacterial density (c.f.u. ml(-1)) calculated. All samples cultured grew Pseudomonas aeruginosa and for two patients Staphylococcus aureus additionally grew for all samples. There was no significant difference in P. aeruginosa density in samples processed at 1, 2, 4 and 6 h following expectoration [8.2 (7.8-8.3) c.f.u. ml(-1), 8.0 (7.8-8.3) c.f.u. ml(-1), 8.0 (7.9-8.2) c.f.u. ml(-1), 8.1 (7.9-8.2) c.f.u. ml(-1), respectively, P=0.392]. Storage for 24 and 48 h at 4 degrees C did not significantly change the bacterial load compared with processing at 1 h [8.03 (7.6-8.2) c.f.u. ml(-1), P=0.07, and 7.96 (7.49-8.22) c.f.u. ml(-1), P=0.09, respectively]. Storage for 24 and 48 h at -20 degrees C significantly reduced P. aeruginosa density [7.1 (6.1-7.7) c.f.u. ml(-1), P=0.005, and 6.9 (6.2-7.6) c.f.u. ml(-1), P=0.008, respectively]. Storage at 25 degrees C for 24 and 48 h was associated with a significant increase in bacterial load [8.3 (8.1-8.6) c.f.u. ml(-1), P=0.009, and 8.4 (8.1-8.5) c.f.u. ml(-1), P=0.03, respectively]. Bacterial density was not affected by storage for up to 6 h following expectoration at 25 degrees C; beyond this, storage at 4 degrees C is preferred.