Variant cell lines that are sensitive to density-dependent inhibition of growth have been isolated from three of four SV40-transformed human fibroblast cell lines. Variants were isolated by plating the transformed cells at low density, by treating them with 5-fluorodeoxyuridine, or by growing them on glutaraldehyde-fixed monolayers of normal cells. The variant cell lines, isolated at a frequency of about 2% of all cells forming colonies after treatment, were initially recognized by colonial morphology, and the variant pheno-types were confirmed, after subculturing, by saturation-density determinations. The variant cell lines reach saturation densities that are 40% or less than those of the parent cell lines, and plate in soft agar medium at reduced efficiency, compared with the parent cells. They retain SV40 T antigen. The modal chromosome numbers of two of the variant cell lines were increased, compared with those of the parent cell lines; two other variants were indistinguishable in chromosome number from the parent cells. Stability of these properties over a 6-month period was demonstrated with two of the variants.