Djungarian hamster cell lines, selected for resistance to 2 microgram/ml of actinomycin D (AD) have been studied. These lines are 1000-4000 times more resistant to AD than the parent cells. AD-resistance is an unstable property. It is lost or diminished when the cells are grown in the absence of AD. The resistant cells show markedly reduced uptake of AD and unrelated agent - colchicin, which indicated that resistance to AD is due to the decrease of plasma membrane permeability. The chromosomal analysis of resistant lines revealed a specific abnormality in their kariotypes, namely, chromosomes containing "homogeneously staining regions" (HSR). These data support the suggestion that AD-resistance is associated with gene amplification.