African bovine trypanosomiasis, caused by the protozoan parasite Trypanosoma congolense, is endemic throughout sub-Saharan Africa and is a major constraint on livestock production. A promising approach to disease control is to understand and exploit naturally evolved trypanotolerance. We describe the first attempt to investigate the transcriptional response of susceptible Boran (Bos indicus) cattle to trypanosome infection via a functional genomics approach using a bovine total leukocyte (BOTL) cDNA microarray platform. Four male Boran cattle were experimentally infected with Trypanosoma congolense and peripheral blood mononuclear cells (PBMC) were collected before infection and 13, 17, 23 and 30 days post-infection (dpi). A reference experimental design was employed using a universal bovine reference RNA pool. Data were normalised to the median of a set of invariant genes (GAPDH) and BRB-Array Tools was used to search for statistically significant differentially expressed genes between each time-point. Using a set of 20 microarray hybridisations, we have made a significant contribution to understanding the temporal transcriptional response of bovine PBMC in vivo to a controlled trypanosome infection. The greatest changes were evident after 13 dpi after parasites were first detected in the blood. Significant differences were observed in clusters of protein kinase C subunits and MHC class I/II related molecules.