Mesophyll protoplasts of Nicotiana tabacum (NR^-/SR^+) and N. glutinosa were electrofused with AC field of 0.5 MHz and 1 kV DC pulse for 2 ms. Fused protoplasts were selected and cultured to the green cell clusters in MSNO_3 medium containing 1.2 ㎍/㎖ streptomycin sulfate. Four plant lines regenerated from selected colonies showed both parental morphological characteristics of leaf and flower and these plant lines were confirmed as somatic hybrids based on electrophoretic patterns of leaf peroxidase. In XhoI restriction patterns of chloroplast DNA, these hybrid plant lines expressed both parent common restriction sites and parent specific sites. One of these hybrid lines exhibited interspecific pattern of both parental chloroplast genomes, indicating nine both parent common sites, one N. tabacum specific site and two N. glutinosa specific sites.