Matrix metalloproteinases form a proteinase family with at least 20 members, which are involved in several pathological conditions and which fulfill a large number of physiological functions. Gelatinase A/MMP-2 is a constitutively produced homeostatic enzyme, whereas gelatinase B/MMP-9 is upregulated in acute and chronic inflammations and forms a target for the development of therapeutic inhibitors. We have used a recently developed assay with fluorescent gelatin to analyze gelatinase inhibitors. A peptidomimetic, based on the consensus sequence of the cleavage sites in type II collagen, and various derivatives of a neutralizing antibody were compared as gelatinase inhibitors. A single-chain variable fragment (scFv) derived from the gelatinase B-selective monoclonal antibody REGA-3G12 was tagged with oligohistidine and was also compared with the untagged scFv. Both scFv derivatives inhibited gelatinase B but the peptidomimetic was inefficient. As an extra control and serendipitously it was found that polyhistidine is an inhibitor of gelatinases, presumably by altering the active site by chelation of the catalytic Zn2+.