Mesophyll protoplasts of N. tabacum. cv. Bright Yellow mutant Aurea and Burley 21 and of N. rustica. cv. Rustica were prepared from leaf tissue by enzymatic digestion and were fused with the aid of polyethylene glycol. When the fused protoplasts were plated on an agar medium or in a liquid medium for cell culture, new cell walls were regenerated from them and the 1st cell division began on the 12th day of culture, and cell division process took place on and after the 21st day. After 1 mo. culture, almost all of the surviving fused protoplasts grew to cell colonies of 1-2 mm in diameter. The cell colonies were subcultured on an agar medium for callus culture. On the medium, they grew vigorously and formed calli of 2 cm in diameter after 2 mo. culture. At this stage, the hybrid calli showing greenish white with a compact appearance were selected in contrast to parental type calli tinged with white or green color. Ten somatic hybrid calli of the combination of Aurea and Rustica and 3 calli of the combination of Burley 21 and Rustica were obtained. Many plantlets were differentiated from these calli when transfered to a medium enriching zeatin. All mature flowering plants were perfectly hybrid, and the shape of the leaves and flowers indicated an intermediate between N. tabacum and N. rustica. The normal hybrids were taller than the parent plant and all hybrid plants were highly resistant to tobacco mosaic virus. Somatic hybrids were aneuploid, having from 60-91 chromosomes. The normal hybrid plants had fertile pollen and seeds and progene were obtained from 4 of the Aurea-Rustica hybrids and 3 of the Burley 21-Rustica hybrids.