Members of the Babesia rap-1 gene family are expressed during multiple parasite stages, and are regulated by both transcriptional and post-transcriptional mechanisms. In all Babesia species, tandemly arranged rap-1 gene copies are separated by an intergervic (IG) region that is hypothesized to regulate gene expression. In this study, we tested that hypothesis by determining whether the Babesia bovis rap-1 IG region could promote extra-chromosomal expression of exogenous genes introduced into merozoites by transfection, and whether a tandem arrangement of IG regions similar to the rap-1 locus enhances exogenous gene expression. Initially, electroporation conditions of B. bovis parasites were determined using expression of the reporter luciferase gene. Both B. bovis transfected by electroporation and Escherichia coli transformed with plasmid p40-15-luc containing the luciferase gene under the control of the B. bovis rap-1 IG and 3(l) flanking regions were able to express luciferase, indicating that the rap-I IG region contains a functional promoter. The chromosomal organization of the B. bovis rap-1 locus includes two identical rap-1 open reading frames and IG regions in a head to tail orientation. To determine whether this orientation enhanced expression of exogenous genes, plasmid constructs containing two rap-I-IG regions controlling expression of the luc and human dihydrofolate reductase (hdhfr) genes, and oriented either in head to head (pLuc-H- 13) or head to tail (pLuc-H- 18) arrangement, were compared. The head to tail orientation of the gene cassettes resulted in a significant increase in the level of luciferase as compared to either head to head orientation or a single IG region construct (p40-15-luc). Thus, an organization that mimics the native structure of the rap-1 locus results in enhanced luciferase expression. These results are the first to demonstrate exogenous gene expression in B. bovis after transfection, and to confirm that the B. bovis rap-1 IG region can promote extra-chromosomal gene expression in vivo. (C) 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.