Lung antiprotease activity is routinely assayed in the supernatant of bronchoalveolar lavage fluid (BALF). In this study the cellular fraction of lavages was also analyzed. Functionally active acid-resistant inhibitors with molecular masses characteristic of the mucus proteinase inhibitor (MPI, 14 kDa) and elastase-specific inhibitor (ESI, 7 kDa) were demonstrated by gel chromatography. Immunocytochemical studies of cells obtained at various postnatal time points from lavages of 10 premature infants with chronic lung disease showed that the inhibitors were confined to neutrophils and macrophages. At each time point, about 70% and 21% of these cells, respectively, stained positively. The polyclonal antibodies usually used to detect MPI did not distinguish between MPI and ESI. Because of this cross reactivity, it was not possible to differentiate between MPI and ESI. Analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) of cells from lavages and of nucleated cells isolated from the peripheral blood showed the production of ESI only, but not of MPI. Nevertheless, MPI can associate with neutrophils and macrophages, as was shown in binding studies with the recombinant protein. These data suggest that when assaying bronchoalveolar lavages (BALs) for these antiproteases in the supernatant only, the total pool of inhibitors may be underestimated.