In previous studies, fusions of transformed X nontransformed CHEF cells have produced hybrids that were suppressed for transformed traits and for tumor formation. During subsequent growth, the suppressed phenotypes were lost coincident with chromosome loss, and in one study the loss of anchorage dependence was correlated with loss of chromosome 1. In this paper, suppression of serum and anchorage requirements for growth is examined with the use of double-mutant tester stocks. Nontumorigenic low serum mutants from CHEF/18 cells are shown to complement with the lowered serum requirement of CHEF/16, a tumorigenic line, indicating that the high serum requirement is dominant and regulated by at least two genes. Similar results were previously reported for the anchorage requirement. Suppression of the two traits is found to segregate independently in hybrid subclones with reduced chromosome numbers, showing that different genes control suppression of the serum and anchorage requirements. Evidence for two modes of suppression, by dominant alleles of transformation genes and by unrelated genes, is presented and discussed.