OBJECTIVE To evaluate the effect of assay buffer ionic strength on assessment of the antielastase activities in bronchoalveolar lavage fluid. METHODS An improved assay protocol in which elastase (in Tris-HCI buffer) is added to increasing volumes of test samples (made up to equal volume with phosphate-buffered saline) was used. RESULTS The percent NE activity inhibited by BALF decreased with increasing NaCl concentration of the buffer. Inhibition of pancreatic elastase (PE) was not affected. One hundred percent inhibition of NE by pure AAT and SLPI standards occurred at molar ratios of 0.91 +/- 0.03 for AAT-to-NE and 0.83 +/- 0.02 for SLPI-to-NE when assayed in buffer with < or = 0.15 mol NaCl/L, compared to ratios of 0.99 +/- 0.02 and 1.06 +/- 0.02, respectively, for assays in buffers with 0.50-1.00 mol NaCl/L (p < 0.05 for AAT-to-NE; p < 0.02 for SLPI-to-NE). The AAT-to-PE molar ratio at 100% inhibition of PE was not affected. Assays in buffer with < or = 0.15 mol NaCl/L indicated that 86.9 +/- 4.1% of AAT and 100.9 +/- 4.9% of SLPI in BALF were active against NE, while assays in buffer with 0.50-1.00 mol NaCl/L showed that 84.4 +/- 3.5% of AAT and 81.6 +/- 5.9% of SLPI present were active. AAT inhibited NE and PE equally only in buffer with 0.50-1.00 mol NaCl/L. CONCLUSIONS The results of assays of BALF antielastase activities depend on the assay buffer NaCl concentration, which may account for the conflicting reports in the literature. The buffer with 0.50-1.00 mol NaCl/L appear to be optimal for valid quantitation of anti-NE activities in BALF.