Procedures are now available for the exploitation of protoplast technology for the genetic manipu- lation of lettuce and for studies of lettuce/virus interactions. These techniques have made it possible to overcome interspecific barriers by protoplast fusion. Following the procedures we developed, we were able to obtain hybrids between Lactuca sativa and two wild Lactuca species (L. tatarica and L. perennis). Moreover, protoplast technology can overcome some of the limitations in the application of mutation techniques in crops. In this paper, we demonstrated the feasibility and the power of our in vitro techni- ques, in combination with induced mutations, for creating new variability in lettuce. Furthermore, we adapted protocols that can provide the opportunity to start studies on mechanisms associated with the different LMV-resistance genes at the cellular level. It is presently possible to obtain protoplasts from different Lactuca species containing different resistance genes. A protocol for specific staining by immu- nofluorescence of infected lettuce protoplasts is described and preliminary electroporation experiments in some Lactuca protoplasts were performed.