Mutants of SV40-transformed mouse fibroblasts have been isolated that have greatly increased cell-substratum adherence. The adherent phenotype (COL-) is recessive, and all mutants analyzed belong to one complementation group. No consistent qualitative differences between wild-type and mutant cells were found with respect to the protein content of the substrate-attached material (SAM), a cell surface fraction left after removal of cells from the substrate with a gentle Ca2+-chelating agent. However, the mutants yielded 2.5-10-fold more SAM than the parental cell line, and the SAM deposited by mutants was able to mediate attachment of transformed cells to a much greater degree than was the SAM from the parental cell line. The mutation, which appears to control the generation of footpads, was shown to cosegregate with resistance to the drug 6-thioguanine, which suggests X-linkage.