Secretory leukocyte protease inhibitor (SLPI) has been proposed as a potential inhibitor of HIV-1 infection in human saliva. Although the ability of recombinant (r) SLPI to inhibit HIV-1 infection of macrophages and primary T-cells has been demonstrated by two independent laboratories, evidence to the contrary has also been reported. This study re-examines the anti-HIV effect of rSLPI and investigates the effects of repeated freeze-thawing and oxidation on the anti-HIV activity of rSLPI. rSLPI inhibited HIV-1BaL infection of human macrophages in a highly variable manner. HPLC and electrospray ionization mass spectrometry (ESI) analyses indicated that variability in our inhibition data could not be attributed to the degradation or oxidation of rSLPI. These results suggest that the variable anti-HIV effect of rSLPI may be due to differential expression of the cell-surface molecule(s) to which SLPI binds rather than to changes in the rSLPI molecule.