Tropical theileriosis is a cattle disease of global economic importance, caused by the tick-borne protozoan parasite Theileria annulata. Conventional control strategies are failing to contain the disease and an attractive alternative is the use of pre-existing genetic resistance or tolerance. However, tropical theileriosis tolerant cattle are less productive than some susceptible breeds. Breeding for combined resistance and production traits requires an understanding of the mechanisms involved in resistance. We have compared the transcriptional response of monocytes derived from tolerant (Sahiwals, Bos indicus) and susceptible (Holstein-Friesians, Bos taurus) cattle to in vitro infection with T. annulata using our recently developed bovine macrophage-specific cDNA microarray. Over 150 genes exhibited breed-specific differential expression during the course of infection, of which nearly one-third were differentially expressed in resting cells, implying that there are inherent differences between monocytes from the two breeds. Fifty sequences currently match only with expressed sequence tags or are unique to the library used to generate the microarray. The greatest breed differences were observed for Toll-like receptor 10 and signal-regulatory protein alpha (SIRPA). Other differentially expressed genes included MHC class II DQ alpha, CD9 and prion protein (PRNP). The differential expression of 40 genes was validated by RT-PCR and a subset of these was validated by quantitative RT-PCR, e.g. PRNP and SIRPA. A large proportion of the differentially expressed genes encode proteins expressed on the plasma membrane or in the extracellular space and cell adhesion was one of the major Gene Ontology biological processes identified. We therefore hypothesise that the dissimilar susceptibility to tropical theileriosis exhibited by Sahiwal and Holstein-Friesian cattle is due to breed-specific differences in the interaction of infected cells with other immune cells, which influences the immune response generated against T. annulata infection.