The subject of the present invention is the construction of multicistronic eukaryotic plasmid expression vectors in which it is possible to express from two to four genes simultaneously and which are characterized by differently regulated bicistronic transcription units. The distinctive characteristic of these vectors is the presence of a CAP-independent translation initiation mechanism which is based on the ability of an IRES (internal ribosomal entry site) sequence to translate two proteins under the control of a single promoter. This family of multicistronic vectors can advantageously be used in various biotechnological applications in whcih the simultaneous expression of two or more genes is necessary, such as gene transfer protocols, DNA-immunization, or for the expression of different molecules in the same cell.