In order to characterize a fibroblast cell line representing normal human skin fibroblasts in three-dimensional cultures, we compared the fibroblast line MSU-1.1, derived from human foreskin and immortalized by v-myc, to primary human dermal fibroblasts (NDF). Our results demonstrate that in contrast to NDF, all MSU-1.1 fibroblasts die within 3–4 d when cultured within three-dimensional contractile collagen matrices. Also, in contrast to NDF, MSU-1.1 cells die markedly in anchored collagen gels as well. Death is due to apoptosis and is attenuated by addition of antibodies against collagen-recognizing receptors α1β1 and α2β1. Apoptosis of NDF in collagen lattices was repressed by an inhibitor of caspase-1, which was ineffective on apoptosis of MSU-1.1. Further, apoptosis by MSU-1.1 fibroblasts was also observed in anchored, i.e., restrained collagen lattices, an environment that supports proliferation of NDF.