Technical Abstract: Babesia bovis is a tick-borne apicomplexan parasite that causes an acute disease in cattle. This study describes stable expression of an exogenous gfp-bsd gene in B. bovis transformed parasites. Cultured B. bovis infected erythrocytes of the biologically cloned Mo7 strain were transfected by electroporation at 1.0kV/.25 microF with either circular or linear plasmids pgfp-bsd-ef or pBS as a control. The plasmid pgfp-bsd-ef was designed to target integration of the gfp-bsd gene to the ef-1alpha locus, which contains two identical gene copies of the ef-1alpha gene. The plasmid was constructed by cloning the firsts 700 bp of the 5’ region of the B. bovis ef-1alpha orf followed by a gfp-bsd fusion gene under the transcriptional control of the B. bovis ef-1alpha promoter and the 3’ region of the rap-1 locus, and 600 bp of the remaining 3’ half of the ef-1alpha orf in the multiple cloning site of a pBS vector plasmid. Cultured transfected B. bovis were selected with blasticidin 24 hours after electroporation. Blasticidin resistant- B. bovis transfected cell lines emerged at different rates only in wells containing pgfp-bsd-ef-transfected parasites, ranging from 8 to 26 days after the start of selection. Four transfected cell lines were selected for further analysis based on their pattern of GFP expression as detected by fluorescence microscopy and high level of resistance to blasticidin. Legitimate integration of the gfp-bsd cassette in the genome was initially demonstrated by Southern blot analysis after chromosome separation by PFGE or on restriction enzyme digested genomic DNA. We are currently characterizing the sites and mechanisms of insertion of the gfp-bsd gene into the B. bovis genome in transfected parasites.