Mesophyll protoplasts of a kanamycin-resistant line of Nicotiana plumbaginifolia were gamma-irradiated and fused with mesophyll protoplasts of N. tabacum plants bearing the sulfur mutation. Hybrid calli were recovered by selection on media containing kanamycin. In one group of experiments, the degree of elimination of donor (N. plumbaginifolia) genetic material in the hybrid calli was assessed by dot-blot hybridization using a N. plumbaginifolia-specific repetitive-DNA sequence as a probe. The elimination of donor DNA was found to increase with increasing gamma dose for all doses tested (5-50 krad). Elimination of donor DNA was also found to continue in the calli for the first 12 months in culture. The degree of chromosome elimination was quite variable; for a 50-krad dose, some hybrids were recovered that retained less than 15% of the donor genome, whereas others retained nearly 50%. In a second set of experiments, the degree of donorchromosome elimination was assessed from the fraction of hybrid calli that exhibited complementation of the Su phenotype due to retention of a wild-type Su allele of the donor. When N. plumbaginifolia protoplasts were inactivated by treatment with iodoacetate, rather than gamma irradiation, all the hybrid calli were green. However, when the donor protoplasts were inactivated by irradiation, the fraction of hybrid calli that were able to complement the Su mutation decreased with increasing gamma dose; for a 50-krad dose only 40% of the hybrid calli were green. From these data, the degree of radiation-induced donor-chromosome elimination was calculated and was found to agree closely with that measured by dot-blot hybridization. We conclude that radiation-induced elimination of donor chromosomes increases with gamma dose and time in culture in N. tabacum (+)N. plumbaginifolia hybrids, but that donor-chromosome elimination is an inherently variable process.