Suppression of tumorigenicity was first shown in hybrids produced by the fusion of a range of different highly malignant tumor cells with diploid fibroblasts. Cytogenetic analysis of these hybrids revealed that suppression involved a genetic region located in one specific chromosome donated to the hybrid cell by the fibroblast parent. The identity of the gene responsible for this dramatic effect has remained obscure. We now present strong evidence that the primary determinant is the gene specifying collagen XV, a proteoglycan closely associated with the basement membrane. We transfected a line of highly tumorigenic human cervical carcinoma cells with an expression vector carrying the full-length cDNA of the human collagen XV gene. We selected clones making various amounts of collagen XV, examined their growth in vitro, and tested their tumorigenicity in nude mice. High levels of collagen XV altered the growth properties of the cells in three-dimensional cultures. Moreover, we found that, in a dose-dependent manner, the production of collagen XV completely suppressed tumorigenicity in clones that synthesized this molecule at high levels. Immunohistologic studies suggest that suppression is associated with extracellular deposition of the proteoglycan at the cell periphery.