The antibiotic blasticidin S inhibits peptide-chain elongation in extracts of bacteria and mammalian cells. After spontaneous or nitrosoguanidine mutagenesis, we have isolated 46 blasticidin S-resistant (Blar) cell lines independently from mouse mammary carcinoma cells (FM3a). Among those Blar clones, we studied two clones, a spontaneously induced one (S501) and a nitrosoguanidine mutagenized one (N742) in more detail. The resistant phenotype of these Blar cells is retained without change for at least four months in the absence of the antibiotic. These Blar cells are 10- to 20-fold more resistant to the cytotoxic action of the antibiotic than their parental cells in vivo. Polyuridylate dependent polyphenylalanine synthesis in vitro with S-30 extracts either from N742 or S501 is 10- to 50-fold more resistant to the inhibitory action of blasticidin S compared to the parental FM3a cells. Ribosomes from FM3a and N742 are fractionated into 40-S and 60-S subunits, and polyphenylalanine synthesis by mixing them in various combinations with S-100 fraction from mouse leukemia L5178Y cells indicating that the resistant phenotype of Blar cells is due to the alteration of 60 S ribosomal subunit. We also found that these two Blar cell lines (N742 and S501) show cross-resistance to gougerotin, puromycin and sparsomycin, but not to emetine or cycloheximide. The polyribosomal pattern of FM3a (Blas) and N742 was compared when the cells were incubated with 3 microgram/ml puromycin for 6 h. Puromycin treatment of Blas cells induced accumulation of monosomes and ribosomal subunits, while little if any transition of polyribosomes into monosome and ribosomal subunits appeared in its counterpart N742 treated with the same dose of puromycin.