Gelatinase B is a regulated matrix metalloproteinase with an important role in the remodelling of extracellular matrices and of basement membranes. To study the structure and function of gelatinase B in the mouse, the cDNA was cloned from a macrophage cell line (WEHI‐3). Using this cDNA, a cosmid clone with the mouse gene was isolated. The complete gene (8 kbp) was sequenced and compared with the human gene structure. There was 78% similarity at the cDNA level and the exon/intron structure of the murine gene was similar to the human counterpart. At the 5′ untranslated side, 1200 bp of the promoter/enhancer region were sequenced and found to contain several trans‐acting‐factor‐binding sites. The mRNA transcription‐initiation site was determined by non‐isotopic primer‐extension analysis. Polymerase‐chain‐reaction amplification of cDNAs yielded indirect evidence for a reverse‐transcription stop in WEHI‐3 cell mRNA. The DNA‐derived mouse‐protein structure exhibited 82% similarity with the human one. This similarity was functionally reflected by cross‐reactivity of the mouse protein with an antiserum against human gelatinase B. The production of murine gelatinase B was studied at the protein level by zymography and at the mRNA level by Northern blot analysis. In WEHI‐3 cells the gelatinase B protein is induced by bacterial lipopolysaccharide, phorbol ester, double‐stranded RNA and the cytokine interleukin‐1. Regulation of activity and structural heterogeneity of gelatinase B in WEHI‐3 cells were shown to occur at the gene regulatory level, by expression of the matrix metalloproteinase inhibitor TIMP‐1, and by glycosylation of the secreted protein.