Hybrid lethality was expressed in seedlings from crosses between Nicotiana nudicaulis and N. tabacum at 28°C. To clarify the subgenome responsible for this phenotype, we crossed N. nudicaulis with the two progenitors of N. tabacum (SSTT), N. sylvestris (SS) and N. tomentosiformis (TT). Seedlings of N. nudicaulis×N. tomentosiformis did not show any lethal symptoms, and these hybrid seedlings subsequently grew to maturity. On the other hand, in the cross N. sylvestris×N. nudicaulis, all seedlings cultured at 28°C died immediately after germination, and others cultured at 34°C and 36°C were nonviable or abnormal; only one hybrid plant survived. These findings suggest that the S subgenome in N. tabacum is responsible for the lethality occurring in hybrids between N. nudicaulis and N. tabacum. Furthermore, to determine whether the Q chromosome induces this hybrid lethality, we crossed a monosomic line of N. tabacum lacking the Q chromosome with N. nudicaulis. Subsequently, we determined the presence or absence of the Q-chromosome-specific DNA marker in hybrid seedlings. Hybrid seedlings both possessing and lacking the Q chromosome showed hybrid lethality when seedlings cultured at 34°C were transferred to 28°C. From these results, we concluded that the Q chromosome is not responsible for the hybrid lethality observed in crosses of N. nudicaulis×N. tabacum.