Interleukin-1alpha (IL-1alpha) is strongly expressed in odontogenic keratocysts. In this study, we investigated the effects of IL-1alpha on the activation of matrix metalloproteinase-2 (MMP-2) in the fibroblasts isolated from odontogenic keratocysts. Odontogenic keratocyst fibroblasts secreted a latent form of MMP-2 (proMMP-2) spontaneously. Type I collagen induced the activation of the proMMP-2, and recombinant human IL-1alpha (rhIL-1alpha) further enhanced the type I collagen-induced activation of proMMP-2 in a dose-dependent manner. The rhIL-1alpha-induced activation of proMMP-2 was inhibited by anti-human IL-1alpha antibody. A reverse-transcription/polymerase chain-reaction (RT-PCR) and Western immunoblotting demonstrated that the expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) mRNA and protein was increased in the fibroblasts when the cells were cultured on type I collagen, and the expression was further enhanced by rhIL-1alpha. Thus, IL-1alpha may up-regulate proMMP-2 activation by increasing the expression of MT1-MMP in the fibroblasts isolated from odontogenic keratocysts synergistically with type I collagen.