Doubly transformed tobacco plants were obtained following sequential transformation steps using two T-DNAs encoding different selection and screening markers: T-DNA-I encoded kanamycin resistance and nopaline synthase; T-DNA-II encoded hygromycin resistance and octopine synthase. A genetic analysis of the inheritance of the selection and screening marker genes in progeny of the doubly tranformed plants revealed that the expression of T-DNA-I genes was often suppressed. This suppression could be correlated with methylation in the promoters of these genes. Surprisingly, both the methylation and inactivation of T-DNA-I genes occurred only in plants containing both T-DNAs: when self-fertilization or backcrossing produced progeny containing only T-DNA-I, expression of the genes on this T-DNA was restored and the corresponding promoters were partially or completely demethylated. These results indicated that the presence of one T-DNA could affect the state of methylation and expression of genes on a second, unlinked T-DNA in the same genome.