Giardia canis virus (GCV) is a double-stranded RNA (dsRNA) virus of the family Totiviridae. In this study, the full length cDNA of the G. canis virus was constructed in pPoly2/sfinot vector and RNA was transcribed in vitro. Virus-free G. canis trophozoites were transfected with in vitro transcribed GCV RNA by electroporation. Transfected trophozoites were cultured for 12, 24, 36, 48, 60, or 72h post transfection for analysis. The ultrastructures of the transfected trophozoites were determined by transmission electron microscopy. The viral particles were detectable sporadically in the cytoplasm as early as 24h post transfection, but became evident and wide-spread 36h post transfection. The number of viral particles increased dramatically from 48 to 60h. Viral particles were released into the culture medium starting at about 60h and detectable in nuclei 72h post transfection. Severe vacuolization was seen in transfected G. canis trophozoites as early as 36h post transfection and persisted throughout the course of this study. The results of the present study indicate that in vitro transcribed GCV transcripts were capable of infecting Giardia trophozoites, apparently replicated and packaged into mature infectious viral particles which were released from the host.