For many studies on matrix metalloproteinases in immunohistochemistry it is important to be able to distinguish between the zymogen and activated forms of the enzymes. Activated human matrix metalloproteinase-9 (MMP-9, gelatinase B) was produced from the proenzyme by limited digestion with trypsin. The products of cleavage were characterised by SDS/PAGE and N-terminal sequencing. Trypsin treatment led to a stepwise removal of the propeptide domain and also caused cleavage within the C-terminal domain. Monoclonal antibodies specific for the activated form of human MMP-9 were raised by using a peptide corresponding to the N-terminus of the activated enzyme as immunogen. The antibodies do not recognise the MMP-9 proenzyme or the active or proenzyme forms of matrix metalloproteinase-2 (MMP-2, gelatinase A) and do not react with unrelated proteins in an unfractionated tissue extract. The antibodies were used to detect, by immunohistochemistry, activated MMP-9 in formalin-fixed, wax-embedded sections from a series of oesophageal cancer cases previously shown to contain MMP-9. All of the tumours contained activated MMP-9 localised to tumour cells and macrophages. As the antibodies are effective in immunohistochemistry on formalin-fixed, wax-embedded sections, they should prove useful for the detection of activated MMP-9 in various disease processes.