Semliki Forest virus (SFV, genus Alphavirus) has a broad host range, high efficiency of viral protein expression, and the ability to stimulate an immune response. These properties have made SFV an attractive tool for development of expression vectors, and plasmid clones containing cDNA of the SFV genome often are used. However, instability of these plasmids resulting from cryptic expression of SFV envelope proteins in Escherichia coli represents a problem both for the development of SFV-based vectors and for SFV research. In this study, an infectious plasmid of SFV, pCMV-SFV4, was constructed; its toxic effect was eliminated by intron insertion in the capsid protein encoding region. When transfected into mammalian cells, the plasmid clone was highly infectious and produced virus with properties identical to those of wild-type SFV. The inserted intron was efficiently and properly removed from the RNA genome of SFV. Therefore, this novel and stabilized infectious SFV plasmid represents a superior tool for basic studies of SFV as well as for biotechnological applications.