Elongation factor 1 alpha (EF-1alpha) is a constitutively expressed, abundant protein that is a key element in eukaryotic protein translation. Because of its high level of transcription, the EF-1alpha promoter has been utilised to drive exogenous gene expression in transfected cells. In this study, we identified and characterised the ef-1alpha locus of Babesia bovis, a causative agent of bovine babesiosis, and examined the transcriptional activity of the EF-1alpha promoter. The ef-1alpha locus in the T2Bo strain of B. bovis contains two identical ef-1alpha genes ('A' and 'B') arranged in a head to head orientation and separated by a 1.4 kb intergenic (IG) region containing a 260 bp terminal inverted repeat. Both ef-1alpha genes encode identical proteins with 448 amino acids and a calculated molecular mass of 49 kDa. While the B. bovis ef-1alpha-IG sequence is conserved among multiple strains of B. bovis, it is not significantly related to any regulatory sequence in the DNA databases. The IG region promotes expression of both ef-1alpha genes. Both fragment Ig-A containing 730 bp upstream of ef-1alpha open reading frame A and fragment Ig-B containing 720 bp upstream of ef-1alpha open reading frame B were able to promote luciferase in transient transfection. In the 5' to 3' orientation, the Ig-B fragment resulted in the highest level of luciferase activity, 10 times higher than positive control plasmid p40-15-luc containing the rap-1 IG region, suggesting that this fragment contains a very strong promoter. Analysis of ef-1alpha transcripts confirms that both ef-1alpha genes are transcribed in merozoites. Interestingly, in contrast to other related intra-erythrocytic apicomplexans, the ef-1alpha locus of B. bovis contains a 160 bp intron in the 5' untranslated region.